Personalized myeloma detection

ABSTRACT

Disclosed herein is a personalized method for monitoring a condition or disorder associated with antibody production in a subject. The method can involve treating a biological sample comprising immunoglobulin from the subject to enzymatically cleave a target immunoglobulin associated with the PCD into one or more variable domain peptide fragments of the target immunoglobulin, and then measuring the one or more variable domain peptide fragments in the sample by quantitative mass spectrometry to quantify the amount of the target immunoglobulin in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/946,629, filed Feb. 28, 2014, which is hereby incorporated herein byreference in its entirety.

BACKGROUND

Multiple myeloma (MM) is a plasma cell cancer characterized by bonemarrow clonal plasmacytosis, monoclonal immunoglobulin expression in theserum and/or urine, lytic bone lesions, hypercalcemia, anemia, and renalfailure. MM patients initially respond to therapy, but relapse withdrug-resistant disease. Therefore, early detection and effectivemonitoring are critical for management of MM patients. Current clinicalassays focus on detection and quantification of the monoclonalimmunoglobulin (or M-protein) secreted by the tumor cells, which isessential for diagnosis and monitoring of patients with MM and otherplasma cell dyscrasias (PCD). The Durie and Salmon staging system andthe International Staging System (ISS) are based on the correlationbetween the expression of the monoclonal immunoglobulin and the diseaseburden. The International Myeloma Working Group guidelines also describethe assessment of treatment outcomes based on the changes in expressionof the M-protein. Patient monitoring strategies present significantchallenges, particularly in the diagnosis of premalignant monoclonalgammopathy of undetermined significance (MGUS), prediction ofprogression from MGUS to MM, assessment of response to therapy, anddetection of relapse.

Evaluation of disease is accomplished by serial measurements of theM-protein in serum and urine using a variety of techniques (Berth M, etal Clin Chem 1999, 45, 309-10; French MAH, et al Clin Exp Immunol 1984,56, 473-5; Haraldsson A, et al Ann Clin Biochem 1991, 28, 461-66; ChenK, et al Nephrology 2005, 10, 594-596; Buckley R, et al J Clin Invest1975, 55, 157-65; Hunder G, et al Arthritis Rheum 1974, 17, 955-63).Typically, initial measurements are made using serum proteinelectrophoresis (SPEP), which is limited in sensitivity to approximately0.1 gram per deciliter (g/dl) (O'Connell T X, et al Am Fam Phys 2005,71, 105-112). The monoclonal immunoglobulin produced in highconcentration by MM cells can be visualized as a narrow, discrete, darkband usually in the γ region of the gel or electropherogram. SPEPdensitometry and total serum protein concentration are used to estimatethe amount of the immunoglobulin secreted by the tumor. Patients can befurther characterized using immunofixation electrophoresis (IFE). IFEscreens test for immunoglobulin G, A, and M heavy chains, as well askappa (κ) and lambda (λ) light chains. Immunoglobulin D and E myelomasare rare; when suspected, IFE is repeated to detect IgD or IgE. Thecombination of SPEP and IFE establishes an estimated level in the serumand type of the immunoglobulin that is secreted by the tumor. Thesetraditionally gel-based techniques have recently been replaced bycapillary array instruments (Jolliff C R, et al Electrophoresis 1997,18, 1781-4). For immunoglobulin heavy chains with high expression, SPEPis the current clinical standard for detecting tumor burden, because thedisease-specific immunoglobulin is directly monitored.

However, several factors limit SPEP in monitoring tumor burden inpatients (Morita K, et al Clin Lab 2004, 50, 415-18; Schreiber W E, etal Am J Clin Pathol 1992, 97, 610-13). Therefore, quantification of theinvolved immunoglobulin by nephelometry is also used to monitor tumorburden (Clark R, et al Electrophoresis 1998, 19, 2479-84), and it hasparticular value for immunoglobulins with lower abundances in serum(e.g. IgD and IgE), particularly because the background expression ofthese immunoglobulins is low. Serum free light chain assays (SFLC) arealso implemented using nephelometry to provide an expression ratiobetween the light chains, which supplements other techniques for thedetection of light chain only disease (Jagannath S Clin Lymphoma Myeloma2007, 7, 518-23; Tate J R, et al Clin Chim Acta 2007, 376, 30-6; Pika T,et al Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2008, 152,61-4). Antibody-based methods for protein quantification are alsoinfluenced by the complexity of the immunoglobulin system of thebiologically derived antiserum and variation in its reactivity, as wellas changes in the levels of proteins in the standard reagents (typicallypooled serum) (Reimer C B, et al Clin Chem 1976, 22, 577-82). Thepresence of immunological subclasses (i.e. IgG1-4 and IgA1-2) also addsto the complexity of the analysis (Reimer C B, et al Clin Chem 1976, 22,577-82).

Minimal residual disease (MRD) is the name given to small numbers ofleukaemic cells that remain in the patient during treatment, or aftertreatment when the patient is in remission. It is the major cause ofrelapse in cancer and leukaemia. However, conventional SPEP is notsufficiently effective for detecting MRD.

A quantitative proteomic assay personalized for an individual patientand useful for direct measurement of the disease-specific immunoglobulinwould significantly increase the sensitivity of detection over SPEP andallow for detection of MRD so treatment can be altered.

SUMMARY

Disclosed herein is a personalized method for monitoring a condition ordisorder associated with antibody production in a subject. Inparticular, the method can be used to monitor a plasma cell dyscrasia(PCD) in a subject. The method can involve treating a biological samplecomprising immunoglobulin from the subject to enzymatically cleave atarget immunoglobulin associated with the PCD into one or more variabledomain peptide fragments of the target immunoglobulin. The method canfurther involve measuring the one or more variable domain peptidefragments in the sample by quantitative mass spectrometry to quantifythe amount of the target immunoglobulin in the sample. In someembodiments, PCD is monitored in the subject by the amount of the targetimmunoglobulin in the sample.

The disclosed method is personalized in that variable domain peptidefragments unique to the specific target immunoglobulin associated withPCD in the subject can be used. Therefore, in some embodiments, themethod first involves identifying variable domain peptide fragments thatcan be used to quantify the target immunoglobulin in the subject. Thismethod can involve determining the amino acid sequence of the targetimmunoglobulin and then identifying in silico one or more variabledomain peptide fragments formed by enzymatic digestion of the targetimmunoglobulin that can be used in the disclosed methods. For example,these peptides preferably contain an amino acid sequences from thevariable domain of the target immunoglobulin that is sufficiently uniqueto distinguish the target immunoglobulin from other immunoglobulin inthe biological sample. For example, the one or more variable domainpeptides fragments can contain the complementarity defining region (CDR)of the target immunoglobulin. In addition, the variable domain peptidesshould also be detectable by a mass spectrometer.

The amino acid sequence of the target immunoglobulin can be determinedeither from the immunoglobulin protein, e.g., isolated from the blood orurine, or from mRNA isolated from isolated plasma cells, e.g., from thebone marrow. In some embodiments, the method involves RNA-sequencingimmunoglobulin mRNA from a plasma cell of the subject that is associatedwith the PCD. This RNA sequence can then be in silico translated intoprotein to determine the amino acid sequence. In some embodiments, 5′rapid amplification of cDNA ends (RACE) is used to sequence the targetimmunoglobulin. Primers can be developed against the constant region ofthe immunoglobulin (toward the C-terminus of the protein) so that thevariable region can then be sequenced by reading back to the 5′ end ofthe RNA.

The disclosed method can be used to detect any class of immunoglobulininvolved in a PCD. For example, the variable domain peptides can be froma human heavy chain of IgG1-4, IgA1-2, IgM, IgD, or IgE. The variabledomain peptides can be from a human kappa light chain or any of thehuman lambda light chain isoforms. In some cases, the immunoglobulin inthe sample is denatured prior to enzymatic cleavage. For example, theimmunoglobulin can be denatured by heat, treatment with urea, disulfidereduction, and/or cysteine alkylation.

In some embodiments, the method further involves a treating step toisolate the target immunoglobulin prior to enzymatic cleavage. Forexample, the treating step can involve one or more of size exclusionchromatography, gel electrophoresis, and/or affinity chromatography.

The immunoglobulin in the sample can be enzymatically cleaved byproteolytic enzyme digestion. Non-limiting examples of proteolyticenzymes include trypsin, pepsin, endoproteinase Lys-C, chymotrypsin,endoproteinase Glu-C, endoproteinase Asp-N, and endoproteinase Arg-C,and any combination thereof.

In some embodiments, the one or more variable peptide fragments aremeasured by spiking in during the mass spectrometry a known amount ofthe one or more variable peptides containing a specific label. Forexample, the specific label can be a heavy isotope label or an aminoacid substitution sufficient to create a detectable mass difference.Non-limiting examples of heavy isotope labels include ²H, ¹³C, and ¹⁵N.The amino acid replacements are preferably conservative substitutions,e.g. leucine to valine or aspartic acid to glutamic acid.

Also disclosed herein are methods for quantifying total immunoglobulinin the biological sample by detecting constant domain peptides fragmentscontaining amino acid sequences from a constant domain of theimmunoglobulin that are conserved within each class of immunoglobulin.In some embodiments, the method involves determining the ratio of targetimmunoglobulin (i.e., variable domain peptides) to total immunoglobulin(i.e., constant domain peptides) in the biological sample. This ratioidentifies the amount of normal immunoglobulin, which can be used todetect immune paresis.

The mass spectrometry of the disclosed methods preferably involves theuse of liquid chromatography coupled to multiple reaction monitoring(LC-MRM). For rapid sample analysis, direct infusion-MRM could also beused. Parallel reaction monitoring and pseudo-MRM could also be used toidentify and quantify the peptides using fragment ions observed intandem mass spectrometry. The mass spectrometry can be conducted on atriple quadrupole mass spectrometer. Ion trap, orbital ion trap, andhybrid quadrupole-time-of-flight mass spectrometers could also be used.

The plasma cell dyscrasia (PCD) can be any plasma cell cancer thatresults in overproduction of an immunoglobulin. Non-limiting examples ofPCDs include multiple myeloma, monoclonal gammopathy of undeterminedsignificance (MGUS), solitary plasmacytoma of bone, extramedullaryplasmacytoma, Waldenström's macroglobulinemia (WM), primary amyloidosis,light chain deposition disease and heavy-chain disease. For example,where the PCD is a multiple myeloma, the plasma cells used to sequencethe target immunoglobulin can be CD138⁺ cells, e.g., extracted from bonemarrow aspirates.

The spectrum of MGUS, solitary plasmacytoma of bone, and asymptomaticand symptomatic multiple myeloma may actually represent a naturalprogression of the same disease. The disclosed methods may be especiallyuseful to monitor this progression, by precise measurement of theincreases in signal/amount of the immunoglobulin secreted by the tumorcells.

The methods may also be used to monitor the efficacy of a treatmentregimen on a subject with a PCD, such as multiple myeloma or MGUS, byevaluating the decrease of the signal/amount for the immunoglobulinspecific to the disease. This can also be used to detect and eliminateminimal residual disease (MRD) since it can detect antibodies from tumorcells at levels approximately 67 times lower than conventional methods.

In addition, these methods could be used for more sensitive detection ofrelapse, which would open earlier therapeutic windows and could lead tomore effective intervention and better patient outcomes. In someembodiments, the methods involve selecting an alternative therapy ifrelapse is detected.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1: Box plots Comparing Monoclonal Immunoglobulin Expression inSerum for MM Patients with IgG, IgA, and IgM Diagnoses to OtherPatients. Patients with positive diagnosis for specific immunoglobulintypes, IgG (A), IgA (B), and IgM (C), are compared with other patients,including both patients not diagnosed with MM and patients diagnosedwith MM that express other types of monoclonal immunoglobulins. Data arenot included for IgD (n=5) and IgE (n=1), because the separation isclear between those patients and the rest of the population. Currentclinical measurements are not able to determine the expression of IgG(or IgA) isoforms, but specific peptides from each sequence can bemonitored by LC-MRM to differentiate the isoforms of these heavy chains.Patients with each IgG subtype, IgG1 (D) and IgG3 (E) are compared withall other patients in the study. Data are not included for IgA2 (n=0),IgG2 (n=2), or IgG4 (n=1), because the separation is clear between thepatients expressing those isoforms and the rest of the population. Ineach plot, dashed lines indicate the maximum normal immunoglobulinlevel.

FIG. 2: Correlation of Immunoglobulin Measurements by Nephelometry andLC-MRM Quantification of Peptides Selected from the Constant Region ofEach Immunoglobulin. Data from patients whose MM secrete eachimmunoglobulin (black diamonds) and from other types of patients (opencircles) are plotted for IgG (A), IgA (B), and IgM (C) measurements.Insets show values for LC-MRM quantification for samples that were belowthe threshold for the nephelometry measurements. LC-MRM could alsoquantify immunoglobulin expression levels that were not detectable bynephelometry; data are presented for IgG (D), IgA (E), and IgM (F).

FIG. 3: LC-MRM Analysis of Total Light Chain Expression in Serum. Boxplots are shown to compare patients expressing kappa light chain intheir monoclonal to other patients (A) and those expressing lambda lightchain to other patients (B). Average κ:λ ratios determined fromtriplicate LC-MRM measurements are also shown for each patient (C), withthe exception of 5 patients with κ monoclonals that produced detectable,but not quantifiable, amounts of λ light chain. The normal ratio fortotal expression of light chains in healthy adults (1.3-2.7) is outlinedby dotted lines in the inset (C); three patients with low monoclonalimmunoglobulin expression had ratios on the opposite side of the normalrange from the light chain indicated in their diagnosis.

FIG. 4: LC-MRM and Nephelometry Have Similar Sensitivity for Detectionof Treatment Response in an IgE MM Patient. The patient was monitoredover time with SPEP (A-C) as well as nephelometry and LC-MRM (D)starting at the time of diagnosis and continuing through the treatmentregimen of a clinical trial combining a proteasome inhibitor and animmunomodulator. Both SPEP (circle) and LC-MRM (diamonds) results showIgE elevation at diagnosis (A). During treatment (B and C), the SPEP isnegative, but elevated IgE is still detected by nephelometry (squares)and LC-MRM (D, letters A-C indicate quantification from the same serumsamples as the SPEP data panels above) are still able to detect elevatedIgE. Nephelometry and LC-MRM can observe the decrease in tumor burdenafter the 5^(th) cycle of treatment, and IgE is not detected (ND) in thepatient serum by either method after the 6^(th) cycle of therapy. Tumorburden is reduced by three orders of magnitude, before theimmunoglobulin is no longer detected.

FIG. 5: RNA-Seq, Protein Sequence, and LC-MRM Verification of Constantand Variable Region Peptides for H929 MM Cells. RNA-seq and in silicotranslation produced a protein sequence, which was verified by LC-MRMdetection of constant (C1-C3) and variable region (V1-V6) peptides fromdigests of conditioned media (A). Spiked H929 Igκ could also be detectedin control serum (B).

FIG. 6: Detection of Peptides from the Disease-Specific Igκ Light Chain.Three peptides derived from RNA-seq and κ light chain assembly usingtumor cells from Patient 1 were observed with LC-MRM in a serum samplewith 0.2 g/dl M-protein: VTITCR (SEQ ID NO:23) (FIG. 6A),SLIYAASSLQSGVPSK (SEQ ID NO:24) (FIG. 6C), and ANQDITNSLVWFQQK (SEQ IDNO:25) (FIG. 6D). Data are provided from Patient 2 to illustrate theuniqueness of the three peptides (FIG. 6B, FIG. 6D, and FIG. 6F,respectively).

FIG. 7: Exemplary workflow for personalized detection of multiplemyeloma tumor burden. Each immunoglobulin (Ig) has unique sequences inthe complementarity defining region (black bars), which will be uniqueto the tumor (A). Two methods are shown for defining the Ig sequence:RNA sequencing and de novo peptide sequencing. Then assays developed forpatients are used for longitudinal monitoring of tumor burden.

DETAILED DESCRIPTION

Liquid chromatography-multiple reaction monitoring mass spectrometry(LC-MRM) using stable isotope dilution has enabled the assessment ofprotein biomarkers (Barr J R, et al Clin Chem 1996, 42, 1676-82;Barnidge D R, et al Anal Chem 2003, 75, 445-51; Gerber S A, et al ProcNatl Acad Sci USA 2003, 100, 6940-45; Anderson L, et al Mol CellProteomics 2006, 5, 573-88; Kuzyk M A, et al Mol Cell Proteomics 2009,8, 1860-77; Keshishian H, et al Mol Cell Proteomics 2007, 6, 2212-29;Kirsch S, et al J Chromatogr A 2007, 1153, 300-6; Kuhn E, et alProteomics 2004, 4, 1175-86; Barnidge D R, et al J Proteome Res 2004, 3,644-52; Yocum A K, et al Proteomics 2010, 10, 3506-14). In addition,collaborative groups have standardized LC-MRM assays at multiple sites(Addona T A, et al Nat Biotechnol 2009, 27, 633-41; Prakash A, et al JProteome Res 2010, 9, 6678-88). Based on these advances, this technologyholds great promise for patient assessment, and LC-MRM is being used intranslational research programs (Koomen J M, et al Mol Cell Proteomics2008, 7, 1780-94). This technology has also been used to measureclinically-relevant protein biomarkers, including troponin I andinterleukin-33 (Kuhn E, et al Clin Chem 2009, 55, 1108-17),apolipoproteins (Agger S A, et al Clin Chem 2010, 56, 1804-13), andthyroglobulin (Hoofnagle A N, et al Clin Chem 2008, 54, 1796-804).

Quantification of immunoglobulins can be achieved at two levels.Peptides from the constant regions can be quantified to evaluate levelsof total immunoglobulin expression. The comparison of LC-MRM of constantregion peptides to immunoglobulin quantification with current clinicaltechniques provides information about the utility, advantages, anddisadvantages of the technique. In addition, development of assays forpeptides from the variable region enables a measurement of thedisease-specific immunoglobulin, similar in specificity to SPEPdetection. Using the strategy of informing proteomics withRNA-sequencing (Evans V C, et al Nat Methods 2012, 9, 1207-11), data areprovided for personalized detection of myeloma tumor burden usingRNA-sequencing and LC-MRM variable region peptide detection for both anin vitro system (i.e. H929 cells) and patients.

Disclosed is a method for detecting and quantifying the tumor burden inpatients with a PCD, such as multiple myeloma. RNA sequencing from tumorcells can be used to define the sequence of the immunoglobulin proteinthat they secrete. This protein can be measured in blood or urine toassess the patient. The disclosed methods increase sensitivity fordetermining the tumor burden in patients with a PCD, such as multiplemyeloma. These measurements can better define the response to therapyand detect relapse earlier.

In current clinical practice, the presence of monoclonal immunoglobulinis detected or quantified by serum protein electrophoresis (SPEP) andthe total amount of the immunoglobulin is quantified by nephelometry. Inreaction monitoring mass spectrometry, specific structural fragments areisolated from specific peptide precursors and quantified by theintegration of their peak area. Each precursor and fragment pair istermed a transition. Several transitions detected at the same timeprovide confidence in the quantification of the molecule.

A level of antibody can, for example, be determined by comparing to areference standard of known values. A person of skill in the art iscapable of preparing a reference standard of known values. The referencestandard can be prepared at the same time, prior to, or afterdetermination of the level of antibody. For MRM mass spectrometry, thesestandards are typically stable-labeled peptides but peptides that arestructurally analogous to the sequence of interest can also be used ifstable-labeled peptides are not available. The signals for theendogenous (biological) peptide are compared to the same set of signalsfrom the standard peptide to enable quantification.

As used herein a control can comprise a known value or reference sample.A known value refers to a value from a diseased sample or a group ofdiseased samples, which can represent, a sample from a subject diagnosedwith cancer or a disease associated with antibody production.Optionally, the reference sample is from a diseased subject of similarsize, weight, height and gender, as the subject being tested or a pooledsample from multiple healthy controls.

Optionally, the antibody comprises a heavy chain selected from the groupconsisting of IgG1-4, IgA1-2, IgM, IgD, and IgE. Optionally, theantibody comprises a light chain selected from a kappa light chain or alambda light chain.

Constant Region Peptides

Exemplary peptides for LC-MRM quantification of total immunoglobulin orspecific isoforms are provided below. These measurements can be pairedwith the detection of specific sequences from the disease-specificmonoclonal immunoglobulin to understand disease burden and assist indetecting relapse if another tumor cell secreting a differentimmunoglobulin grows out after treatment. All peptides listed below caninclude possible alkylation of cysteine with iodoacetamide (orcomparable reagents) and methionine oxidation. In addition, peptideswith missed cleavages could be detected, which would be made ofcombinations of the sequences below.

IgG1 Peptides from Constant Region Trypsin: (SEQ ID NO: 26)GFYPSDIAVEWESNGQPENNYK, (SEQ ID NO: 27) TPEVTCVVVDVSHEDPEVK,(SEQ ID NO: 28) TTPPVLDSDGSFFLYSK, (SEQ ID NO: 29) VVSVLTVLHQDWLNGK,(SEQ ID NO: 30) FNWYVDGVEVHNAK, (SEQ ID NO: 31) EPQVYTLPPSR,(SEQ ID NO: 32) STSGGTAALGCLVK, (SEQ ID NO: 33) EEQYNSTYR,(SEQ ID NO: 1) GPSVFPLAPSSK, (SEQ ID NO: 34) NQVSLTCLVK, (SEQ ID NO: 35)ALPAPIEK, (SEQ ID NO: 36) DTLMISR, (SEQ ID NO: 37) SLSLSPGK; Lys-C:(SEQ ID NO: 38) GFYPSDIAVEWESNGQPENNYK, (SEQ ID NO: 39)THTCPPCPAPELLGGPSVFLFPPK, (SEQ ID NO: 40) GQPREPQVYTLPPSRDELTK,(SEQ ID NO: 41) TTPPVLDSDGSFFLYSK, (SEQ ID NO: 42) FNWYVDGVEVHNAK,(SEQ ID NO: 43) STSGGTAALGCLVK, (SEQ ID NO: 44) GPSVFPLAPSSK,(SEQ ID NO: 45) NQVSLTCLVK, (SEQ ID NO: 46) ALPAPIEK, (SEQ ID NO: 47)SLSLSPGK; Arg-C: (SEQ ID NO: 48) EPQVYTLPPSR, (SEQ ID NO: 49) EEQYNSTYR;Chymotrypsin: (SEQ ID NO: 50) ISRTPEVTCVVVDVSHEDPEVKF, (SEQ ID NO: 51)VDGVEVHNAKTKPREEQY, (SEQ ID NO: 52) APSSKSTSGGTAAL, (SEQ ID NO: 53)ESNGQPENNY, (SEQ ID NO: 54) TSGVHTFPAVL, (SEQ ID NO: 55) ASTKGPSVFPL,(SEQ ID NO: 56) YPSDIAVEW, (SEQ ID NO: 57) SSVVTVPSSSL, (SEQ ID NO: 58)FPEPVTVSW, (SEQ ID NO: 59) FPPKPKDTL, (SEQ ID NO: 60) TLPPSRDEL,(SEQ ID NO: 61) TVDKSRW, (SEQ ID NO: 62) TKNQVSL, (SEQ ID NO: 63)KTTPPVL; Glu-C (phosphate buffer): (SEQ ID NO: 64) QYNSTYRVVSVLTVLHQD,(SEQ ID NO: 65) LTKNQVSLTCLVKGFYPSD, (SEQ ID NO: 66) KSRWQQGNVFSCSVMHE,(SEQ ID NO: 67) ALHNHYTQKSLSLSPGK, (SEQ ID NO: 68) LLGGPSVFLFPPKPKD,(SEQ ID NO: 69) YKCKVSNKALPAPIE, (SEQ ID NO: 70) GSFFLYSKLTVD,(SEQ ID NO: 71) VHNAKTKPREE, (SEQ ID NO: 72) KTHTCPPCPAPE,(SEQ ID NO: 73) NNYKTTPPVLD, (SEQ ID NO: 74) VKFNWYVD, (SEQ ID NO: 75)TLMISRTPE, (SEQ ID NO: 76) KKVEPKSCD, (SEQ ID NO: 77) WLNGKE;Glu-C (bicarbonate buffer): (SEQ ID NO: 78) LTKNQVSLTCLVKGFYPSDIAVE,(SEQ ID NO: 79) ALHNHYTQKSLSLSPGK, (SEQ ID NO: 80) YKCKVSNKALPAPIE,(SEQ ID NO: 81) VKFNWYVDGVE, (SEQ ID NO: 82) VHNAKTKPREE,(SEQ ID NO: 83) VTCVVVDVSHE, (SEQ ID NO: 84) SNGQPE; Asp-N:(SEQ ID NO: 85) DIAVEWESNGQPENNYKTTPPVL, (SEQ ID NO: 86)DELTKNQVSLTCLVKGFYPS, (SEQ ID NO: 87) DTLMISRTPEVTCVVV, (SEQ ID NO: 88)DGSFFLYSKLTV, (SEQ ID NO: 89) DPEVKFNWYV, (SEQ ID NO: 90) DKKVEPKSC;IgG2 Peptides from Constant Region Trypsin: (SEQ ID NO: 91)GFYPSDISVEWESNGQPENNYK, (SEQ ID NO: 92) TTPPMLDSDGSFFLYSK,(SEQ ID NO: 93) VVSVLTVVHQDWLNGK, (SEQ ID NO: 94) STSESTAALGCLVK,(SEQ ID NO: 95) EPQVYTLPPSR, (SEQ ID NO: 96) GPSVFPLAPCSR,(SEQ ID NO: 97) EEQFNSTFR, (SEQ ID NO: 98) NQVSLTCLVK, (SEQ ID NO: 99)DTLMISR, (SEQ ID NO: 2) GLPAPIEK, (SEQ ID NO: 100) SLSLSPGK; Lys-C:(SEQ ID NO: 101) GPSVFPLAPCSRSTSESTAALGCLVK, (SEQ ID NO: 102)GFYPSDISVEWESNGQPENNYK, (SEQ ID NO: 103) CCVECPPCPAPPVAGPSVFLFPPK,(SEQ ID NO: 104) GQPREPQVYTLPPSREEMTK, (SEQ ID NO: 105)TTPPMLDSDGSFFLYSK, (SEQ ID NO: 106) NQVSLTCLVK, (SEQ ID NO: 107)GLPAPIEK, (SEQ ID NO: 108) SLSLSPGK; Arg-C: (SEQ ID NO: 109)ASTKGPSVFPLAPCSR, (SEQ ID NO: 110) EPQVYTLPPSR, (SEQ ID NO: 111)EEQFNSTFR; Chymotrypsin: (SEQ ID NO: 112) ISRTPEVTCVVVDVSHEDPEVQF,(SEQ ID NO: 113) VDGVEVHNAKTKPREEQF, (SEQ ID NO: 114) APCSRSTSESTAAL,(SEQ ID NO: 115) ESNGQPENNY, (SEQ ID NO: 116) TSGVHTFPAVL,(SEQ ID NO: 117) SSVVTVPSSNF, (SEQ ID NO: 118) ASTKGPSVFPL,(SEQ ID NO: 119) YPSDISVEW, (SEQ ID NO: 120) FPEPVTVSW, (SEQ ID NO: 121)TLPPSREEM, (SEQ ID NO: 122) FPPKPKDTL, (SEQ ID NO: 123) TVDKSRW,(SEQ ID NO: 124) TVVHQDW, (SEQ ID NO: 125) TKNQVSL;Glu-C (phosphate buffer): (SEQ ID NO: 126) CPPCPAPPVAGPSVFLFPPK PKD,(SEQ ID NO: 127) MTKNQVSLTCLVKGFYPSD, (SEQ ID NO: 128)QFNSTFRVVSVLTVVHQD, (SEQ ID NO: 129) KSRWQQGNVFSCSVMHE, (SEQ ID NO: 130)ASTKGPSVFPLAPCSRSTSE, (SEQ ID NO: 131) ALHNHYTQKSLSLSPGK,(SEQ ID NO: 132) YKCKVSNKGLPAPIE, (SEQ ID NO: 133) GSFFLYSKLTVD,(SEQ ID NO: 134) VHNAKTKPREE, (SEQ ID NO: 135) NNYKTTPPMLD,(SEQ ID NO: 136) STAALGCLVKD, (SEQ ID NO: 137) VQFNWYVD,(SEQ ID NO: 138) TLMISRTPE, (SEQ ID NO: 139) HKPSNTKVD, (SEQ ID NO: 140)WLNGKE, (SEQ ID NO: 141) RKCCVE, (SEQ ID NO: 142) VTCVVVD;Glu-C (bicarbonate buffer): (SEQ ID NO: 143) MTKNQVSLTCLVKGFYPSDISVE,(SEQ ID NO: 144) ASTKGPSVFPLAPCSRSTSE, (SEQ ID NO: 145)ALHNHYTQKSLSLSPGK, (SEQ ID NO: 146) YKCKVSNKGLPAPIE, (SEQ ID NO: 147)VQFNWYVDGVE, (SEQ ID NO: 148) VHNAKTKPREE, (SEQ ID NO: 149) VTCVVVDVSHE,(SEQ ID NO: 150) RKCCVE; Asp-N: (SEQ ID NO: 151)DISVEWESNGQPENNYKTTPPML, (SEQ ID NO: 152) DTLMISRTPEVTCVVV,(SEQ ID NO: 153) DGSFFLYSKLTV, (SEQ ID NO: 154) DPEVQFNWYV,(SEQ ID NO: 155) DHKPSNTKV; IgG3 Peptides from Constant Region Trypsin:(SEQ ID NO: 156) TPEVTCVVVDVSHEDPEVQFK, (SEQ ID NO: 157)WQQGNIFSCSVMHEALHNR, (SEQ ID NO: 158) CPAPELLGGPSVFLFPPKPK,(SEQ ID NO: 159) VVSVLTVLHQDWLNGK, (SEQ ID NO: 3) WYVDGVEVHNAK,(SEQ ID NO: 160) TPLGDTTHTCPR, (SEQ ID NO: 161) EPQVYTLPPSR,(SEQ ID NO: 162) STSGGTAALGCLVK, (SEQ ID NO: 163) GPSVFPLAPCSR,(SEQ ID NO: 164) EEQYNSTFR, (SEQ ID NO: 165) NQVSLTCLVK,(SEQ ID NO: 166) SCDTPPPCPR, (SEQ ID NO: 167) ALPAPIEK, (SEQ ID NO: 168)DTLMISR, (SEQ ID NO: 169) SLSLSPGK; Lys-C: (SEQ ID NO: 170)GPSVFPLAPCSRSTSGGTAALGCLVK, (SEQ ID NO: 171) GQPREPQVYTLPPSREEMTK,(SEQ ID NO: 172) TPLGDTTHTCPRCPEPK, (SEQ ID NO: 173) SCDTPPPCPRCPEPK,(SEQ ID NO: 174) WYVDGVEVHNAK, (SEQ ID NO: 175) NQVSLTCLVK,(SEQ ID NO: 176) ALPAPIEK, (SEQ ID NO: 177) SLSLSPGK; Arg-C:(SEQ ID NO: 178) WQQGNIFSCSVMHEALHNR, (SEQ ID NO: 179) VELKTPLGDTTHTCPR,(SEQ ID NO: 180) CPEPKSCDTPPPCPR, (SEQ ID NO: 181) ASTKGPSVFPLAPCSR,(SEQ ID NO: 182) FTQKSLSLSPGK, (SEQ ID NO: 183) EPQVYTLPPSR,(SEQ ID NO: 184) EEQYNSTFR; Chymotrypsin: (SEQ ID NO: 185)ISRTPEVTCVVVDVSHEDPEVQF, (SEQ ID NO: 186) TCNVNHKPSNTKVDKRVEL,(SEQ ID NO: 187) VDGVEVHNAKTKPREEQY, (SEQ ID NO: 188) APCSRSTSGGTAAL,(SEQ ID NO: 189) TSGVHTFPAVL, (SEQ ID NO: 190) ESSGQPENNY,(SEQ ID NO: 191) ASTKGPSVFPL, (SEQ ID NO: 192) YPSDIAVEW,(SEQ ID NO: 193) SSVVTVPSSSL, (SEQ ID NO: 194) FPEPVTVSW,(SEQ ID NO: 195) TLPPSREEM, (SEQ ID NO: 196) FPPKPKDTL, (SEQ ID NO: 197)TVDKSRW, (SEQ ID NO: 198) TKNQVSL; Glu-C (phosphate buffer):(SEQ ID NO: 199) MTKNQVSLTCLVKGFYPSD, (SEQ ID NO: 200)QYNSTFRVVSVLTVLHQD, (SEQ ID NO: 201) KSRWQQGNIFSCSVMHE, (SEQ ID NO: 202)ALHNRFTQKSLSLSPGK, (SEQ ID NO: 203) LLGGPSVFLFPPKPKD, (SEQ ID NO: 204)TTHTCPRCPEPKSCD, (SEQ ID NO: 205) YKCKVSNKALPAPIE, (SEQ ID NO: 206)TPPPCPRCPEPKSCD, (SEQ ID NO: 207) GSFFLYSKLTVD, (SEQ ID NO: 208)VHNAKTKPREE, (SEQ ID NO: 209) NNYNTTPPMLD, (SEQ ID NO: 210)TPPPCPRCPAPE, (SEQ ID NO: 211) VQFKWYVD, (SEQ ID NO: 212) TLMISRTPE,(SEQ ID NO: 213) WLNGKE, (SEQ ID NO: 214) LKTPLGD, (SEQ ID NO: 215)VTCVVVD; Glu-C (bicarbonate buffer): (SEQ ID NO: 216)MTKNQVSLTCLVKGFYPSDIAVE, (SEQ ID NO: 217) ALHNRFTQKSLSLSPGK,(SEQ ID NO: 218) YKCKVSNKALPAPIE, (SEQ ID NO: 219) VQFKWYVDGVE,(SEQ ID NO: 220) VHNAKTKPREE, (SEQ ID NO: 221) VTCVVVDVSHE; Asp-N:(SEQ ID NO: 222) DIAVEWESSGQPENNYNTTPPML, (SEQ ID NO: 223)DTLMISRTPEVTCVVV, (SEQ ID NO: 224) DTTHTCPRCPEPKSC, (SEQ ID NO: 225)DTPPPCPRCPEPKSC, (SEQ ID NO: 226) DGSFFLYSKLTV, (SEQ ID NO: 227)DPEVQFKWYV, (SEQ ID NO: 228) DKRVELKTPLG;IgG4 Peptides from Constant Region Trypsin: (SEQ ID NO: 229)GFYPSDIAVEWESNGQPENNYK, (SEQ ID NO: 4) TTPPVLDSDGSFFLYSR,(SEQ ID NO: 230) EPQVYTLPPSQEEMTK, (SEQ ID NO: 231) VVSVLTVLHQDWLNGK,(SEQ ID NO: 232) TYTCNVDHKPSNTK, (SEQ ID NO: 233) STSESTAALGCLVK,(SEQ ID NO: 234) GPSVFPLAPCSR, (SEQ ID NO: 235) EEQFNSTYR,(SEQ ID NO: 236) NQVSLTCLVK, (SEQ ID NO: 237) DTLMISR, (SEQ ID NO: 238)GLPSSIEK, (SEQ ID NO: 239) SLSLSLGK; Lys-C: (SEQ ID NO: 240)GPSVFPLAPCSRSTSESTAALGCLVK, (SEQ ID NO: 241) GFYPSDIAVEWESNGQPENNYK,(SEQ ID NO: 242) TTPPVLDSDGSFFLYSRLTVDK, (SEQ ID NO: 243)GQPREPQVYTLPPSQEEMTK, (SEQ ID NO: 244) NQVSLTCLVK, (SEQ ID NO: 245)TYTCNVDHK, (SEQ ID NO: 246) GLPSSIEK, (SEQ ID NO: 247) SLSLSLGK; Arg-C:(SEQ ID NO: 248) ASTKGPSVFPLAPCSR, (SEQ ID NO: 249) EEQFNSTYR,(SEQ ID NO: 250) LTVDKSR; Chymotrypsin: (SEQ ID NO: 251)ISRTPEVTCVVVDVSQEDPEVQF, (SEQ ID NO: 252) TCNVDHKPSNTKVDKRVESKY,(SEQ ID NO: 253) VDGVEVHNAKTKPREEQF, (SEQ ID NO: 254) APCSRSTSESTAAL,(SEQ ID NO: 255) GPPCPSCPAPEF, (SEQ ID NO: 256) ESNGQPENNY,(SEQ ID NO: 257) TSGVHTFPAVL, (SEQ ID NO: 258) ASTKGPSVFPL,(SEQ ID NO: 259) YPSDIAVEW, (SEQ ID NO: 260) SSVVTVPSSSL,(SEQ ID NO: 261) FPEPVTVSW, (SEQ ID NO: 262) FPPKPKDTL, (SEQ ID NO: 263)TLPPSQEEM, (SEQ ID NO: 264) TVDKSRW, (SEQ ID NO: 265) TKNQVSL,(SEQ ID NO: 266) KTTPPVL; Glu-C (phosphate buffer): (SEQ ID NO: 267)MTKNQVSLTCLVKGFYPSD, (SEQ ID NO: 268) QFNSTYRVVSVLTVLHQD,(SEQ ID NO: 269) ASTKGPSVFPLAPCSRSTSE, (SEQ ID NO: 270)ALHNHYTQKSLSLSLGK, (SEQ ID NO: 271) FLGGPSVFLFPPKPKD, (SEQ ID NO: 272)YKCKVSNKGLPSSIE, (SEQ ID NO: 273) SKYGPPCPSCPAPE, (SEQ ID NO: 274)GSFFLYSRLTVD, (SEQ ID NO: 275) VHNAKTKPREE, (SEQ ID NO: 276)NNYKTTPPVLD, (SEQ ID NO: 277) GNVFSCSVMHE, (SEQ ID NO: 278) STAALGCLVKD,(SEQ ID NO: 279) VQFNWYVD, (SEQ ID NO: 280) TLMISRTPE, (SEQ ID NO: 281)HKPSNTKVD, (SEQ ID NO: 282) KSRWQE, (SEQ ID NO: 283) WLNGKE,(SEQ ID NO: 284) VTCVVVD; Glu-C (bicarbonate buffer): (SEQ ID NO: 285)MTKNQVSLTCLVKGFYPSDIAVE, (SEQ ID NO: 286) ASTKGPSVFPLAPCSRSTSE,(SEQ ID NO: 287) ALHNHYTQKSLSLSLGK, (SEQ ID NO: 288) YKCKVSNKGLPSSIE,(SEQ ID NO: 289) SKYGPPCPSCPAPE, (SEQ ID NO: 290) VQFNWYVDGVE,(SEQ ID NO: 291) VHNAKTKPREE, (SEQ ID NO: 292) GNVFSCSVMHE,(SEQ ID NO: 293) VTCVVVDVSQE; Asp-N: (SEQ ID NO: 294)DIAVEWESNGQPENNYKTTPPVL, (SEQ ID NO: 295) DTLMISRTPEVTCVVV,(SEQ ID NO: 296) DGSFFLYSRLTV, (SEQ ID NO: 297) DPEVQFNWYV,(SEQ ID NO: 298) DHKPSNTKV; IgA1 Peptides from Constant Region Trypsin:(SEQ ID NO: 299) DLCGCYSVSSVLPGCAEPWNHGK, (SEQ ID NO: 300)LAGKPTHVNVSVVMAEVDGTCY, (SEQ ID NO: 301) GDTFSCMVGHEALPLAFTQK,(SEQ ID NO: 302) QEPSQGTTTFAVTSILR, (SEQ ID NO: 303) DASGVTFTWTPSSGK,(SEQ ID NO: 304) TFTCTAAYPESK, (SEQ ID NO: 305) WLQGSQELPR,(SEQ ID NO: 306) SAVQGPPER, (SEQ ID NO: 5) TPLTATLSK, (SEQ ID NO: 307)YLTWASR, (SEQ ID NO: 308) VAAEDWK, (SEQ ID NO: 309) SVTCHVK,(SEQ ID NO: 310) ASPTSPK; Lys-C: (SEQ ID NO: 311) GDTFSCMVGHEALPLAFTQK,(SEQ ID NO: 312) DVLVRWLQGSQELPREK, (SEQ ID NO: 313) PTHVNVSVVMAEVDGTCY,(SEQ ID NO: 314) TFTCTAAYPESK, (SEQ ID NO: 315) TPLTATLSK,(SEQ ID NO: 316) TIDRLAGK, (SEQ ID NO: 317) SVTCHVK; Arg-C:(SEQ ID NO: 318) DASGVTFTWTPSSGKSAVQGPPER, (SEQ ID NO: 319)LAGKPTHVNVSVVMAEVDGTCY, (SEQ ID NO: 320) QEPSQGTTTFAVTSILR,(SEQ ID NO: 321) WLQGSQELPR, (SEQ ID NO: 322) EKYLTWASR,(SEQ ID NO: 323) GFSPKDVLVR; Chymotrypsin: (SEQ ID NO: 324)SESGQGVTARNFPPSQDASGDL, (SEQ ID NO: 325) TPSSGKSAVQGPPERDL,(SEQ ID NO: 326) CSTQPDGNVVIACL, (SEQ ID NO: 327) ASRQEPSQGTTTF,(SEQ ID NO: 328) TCTAAYPESKTPL, (SEQ ID NO: 329) AGKPTHVNVSVVM,(SEQ ID NO: 330) QGSQELPREKY, (SEQ ID NO: 331) SVSSVLPGCAEPW,(SEQ ID NO: 332) AGKSVTCHVKHY, (SEQ ID NO: 333) ASPTSPKVFPL,(SEQ ID NO: 334) TQKTIDRL, (SEQ ID NO: 335) LPPPSEEL, (SEQ ID NO: 336)AEVDGTCY, (SEQ ID NO: 337) RDASGVTF, (SEQ ID NO: 338) TLPATQCL,(SEQ ID NO: 339) RVAAEDW, (SEQ ID NO: 340) VGHEALPL, (SEQ ID NO: 341)RPEVHL, (SEQ ID NO: 342) SKSGNTF, (SEQ ID NO: 343) FPQEPL;Glu-C (phosphate buffer): (SEQ ID NO: 344) GNVVIACLVQGFFPQEPLSVTWSE,(SEQ ID NO: 345) ASGVTFTWTPSSGKSAVQGPPE, (SEQ ID NO: 346)SKTPLTATLSKSGNTFRPE, (SEQ ID NO: 347) ASPTSPKVFPLSLCSTQPD,(SEQ ID NO: 348) RLAGKPTHVNVSVVMAE, (SEQ ID NO: 349) LVTLTCLARGFSPKD,(SEQ ID NO: 350) SGQGVTARNFPPSQD, (SEQ ID NO: 351) ALPLAFTQKTID,(SEQ ID NO: 352) VLVRWLQGSQE, (SEQ ID NO: 353) ANLTCTLTGLRD,(SEQ ID NO: 354) VHLLPPPSEE, (SEQ ID NO: 903) TFSCMVGHE;;Glu-C (bicarbonate buffer): (SEQ ID NO: 355) SKTPLTATLSKSGNTFRPE,(SEQ ID NO: 356) DWKKGDTFSCMVGHE, (SEQ ID NO: 357) VHLLPPPSEE,(SEQ ID NO: 358) DLLLGSE; Asp-N: (SEQ ID NO: 359)DASGVTFTWTPSSGKSAVQGPPER, (SEQ ID NO: 360) DTFSCMVGHEALPLAFTQKTI,(SEQ ID NO: 361) DRLAGKPTHVNVSVVMAEV, (SEQ ID NO: 362)DLLLGSEANLTCTLTGLR, (SEQ ID NO: 363) ASPTSPKVFPLSLCSTQP;IgA2 Peptides from Constant Region Trypsin: (SEQ ID NO: 364)MAGKPTHVNVSVVMAEVDGTCY, (SEQ ID NO: 365) GDTFSCMVGHEALPLAFTQK,(SEQ ID NO: 366) QEPSQGTTTFAVTSILR, (SEQ ID NO: 6) DASGATFTWTPSSGK,(SEQ ID NO: 367) WLQGSQELPR, (SEQ ID NO: 368) TPLTANITK,(SEQ ID NO: 369) SAVQGPPER, (SEQ ID NO: 370) YLTWASR, (SEQ ID NO: 371)VAAEDWK, (SEQ ID NO: 372) SVTCHVK; Lys-C: (SEQ ID NO: 373)GDTFSCMVGHEALPLAFTQK, (SEQ ID NO: 374) DVLVRWLQGSQELPREK,(SEQ ID NO: 375) PTHVNVSVVMAEVDGTCY, (SEQ ID NO: 376) TPLTANITK,(SEQ ID NO: 377) TIDRMAGK, (SEQ ID NO: 378) SVTCHVK; Arg-C:(SEQ ID NO: 379) DASGATFTWTPSSGKSAVQGPPER, (SEQ ID NO: 380)MAGKPTHVNVSVVMAEVDGTCY, (SEQ ID NO: 381) QEPSQGTTTFAVTSILR,(SEQ ID NO: 382) WLQGSQELPR, (SEQ ID NO: 383) EKYLTWASR,(SEQ ID NO: 384) GFSPKDVLVR, Chymotrypsin: (SEQ ID NO: 385)TNPSQDVTVPCPVPPPPPCCHPRL, (SEQ ID NO: 386) SESGQNVTARNFPPSQDASGDL,(SEQ ID NO: 387) TLPATQCPDGKSVTCHVKHY, (SEQ ID NO: 388)TPSSGKSAVQGPPERDL, (SEQ ID NO: 389) DSTPQDGNVVVACL, (SEQ ID NO: 390)ASRQEPSQGTTTF, (SEQ ID NO: 391) AGKPTHVNVSVVM, (SEQ ID NO: 392)QGSQELPREKY, (SEQ ID NO: 393) SVSSVLPGCAQPW, (SEQ ID NO: 394)TANITKSGNTF, (SEQ ID NO: 395) TQKTIDRM, (SEQ ID NO: 396) TCTAAHPEL,(SEQ ID NO: 397) LPPPSEEL, (SEQ ID NO: 398) AEVDGTCY, (SEQ ID NO: 399)RVAAEDW, (SEQ ID NO: 400) VGHEALPL, (SEQ ID NO: 401) RDASGATF,(SEQ ID NO: 402) RPEVHL, (SEQ ID NO: 403) FPQEPL;Glu-C (phosphate buffer): (SEQ ID NO: 404) LCGCYSVSSVLPGCAQPWNHGE,(SEQ ID NO: 405) ASGATFTWTPSSGKSAVQGPPE, (SEQ ID NO: 406)LKTPLTANITKSGNTFRPE, (SEQ ID NO: 407) GKSVTCHVKHYTNPSQD,(SEQ ID NO: 408) LYTTSSQLTLPATQCPD, (SEQ ID NO: 409) RMAGKPTHVNVSVVMAE,(SEQ ID NO: 410) LVTLTCLARGFSPKD, (SEQ ID NO: 411) SGQNVTARNFPPSQD,(SEQ ID NO: 412) ALPLAFTQKTID, (SEQ ID NO: 413) VLVRWLQGSQE,(SEQ ID NO: 414) ANLTCTLTGLRD, (SEQ ID NO: 415) VHLLPPPSEE,(SEQ ID NO: 416) TFTCTAAHPE, (SEQ ID NO: 417) TFSCMVGHE;Glu-C (bicarbonate buffer): (SEQ ID NO: 418) RDLCGCYSVSSVLPGCAQPWNHGE,(SEQ ID NO: 419) LKTPLTANITKSGNTFRPE, (SEQ ID NO: 420) DWKKGDTFSCMVGHE,(SEQ ID NO: 421) VHLLPPPSEE, (SEQ ID NO: 422) TFTCTAAHPE,(SEQ ID NO: 423) DLLLGSE; Asp-N: (SEQ ID NO: 424)DASGATFTWTPSSGKSAVQG PPER, (SEQ ID NO: 425) DTFSCMVGHEALPLAFTQKT I,(SEQ ID NO: 426) DRMAGKPTHVNVSVVMAEV, (SEQ ID NO: 427)DGKSVTCHVKHYTNPSQ, (SEQ ID NO: 428) DLLLGSEANLTCTLTGLR, (SEQ ID NO: 429)DLYTTSSQLTLPATQCP; IgIVI Peptides from Constant Region Trypsin:(SEQ ID NO: 430) STGKPTLYNVSLVMSDTAGTCY, (SEQ ID NO: 431)LTCLVTDLTTYDSVTISWTR, (SEQ ID NO: 432) GLTFQQNASSMCVPDQDTAIR,(SEQ ID NO: 433) GVALHRPDVYLLPPAR, (SEQ ID NO: 434) FTCTVTHTDLPSPLK,(SEQ ID NO: 435) VFAIPPSFASIFLTK, (SEQ ID NO: 436) QVGSGVTTDQVQAEAK,(SEQ ID NO: 437) YVTSAPMPEPQAPGR, (SEQ ID NO: 438) ESDWLGQSMFTCR,(SEQ ID NO: 439) DVMQGTDEHVVCK, (SEQ ID NO: 440) NVPLPVIAELPPK,(SEQ ID NO: 441) YAATSQVLLPSK, (SEQ ID NO: 442) LICQATGFSPR,(SEQ ID NO: 443) QIQVSWLR, (SEQ ID NO: 444) NNSDISSTR, (SEQ ID NO: 8)DGFFGNPR, (SEQ ID NO: 445) VSVFVPPR, (SEQ ID NO: 446) VQHPNGNK,(SEQ ID NO: 447) ESGPTTYK, (SEQ ID NO: 448) VTSTLTIK, (SEQ ID NO: 449)GQPLSPEK, (SEQ ID NO: 450) QTISRPK, (SEQ ID NO: 451) GFPSVLR,(SEQ ID NO: 452) EQLNLR, (SEQ ID NO: 453) QNGEAVK; Lys-C:(SEQ ID NO: 454) LICQATGFSPRQIQVSWLREGK, (SEQ ID NO: 455)NNSDISSTRGFPSVLRGGK, (SEQ ID NO: 456) PTLYNVSLVMSDTAGTCY,(SEQ ID NO: 457) VSVFVPPRDGFFGNPRK, (SEQ ID NO: 458) QVGSGVTTDQVQAEAK,(SEQ ID NO: 459) DVMQGTDEHVVCK, (SEQ ID NO: 460) NVPLPVIAELPPK,(SEQ ID NO: 461) YAATSQVLLPSK, (SEQ ID NO: 462) VQHPNGNK,(SEQ ID NO: 463) ESGPTTYK, (SEQ ID NO: 464) VTSTLTIK, (SEQ ID NO: 465)QTISRPK; Arg-C: (SEQ ID NO: 466) GQPLSPEKYVTSAPMPEPQAPGR,(SEQ ID NO: 467) GLTFQQNASSMCVPDQDTAIR, (SEQ ID NO: 468) KSKLICQATGFSPR,(SEQ ID NO: 469) QIQVSWLR, (SEQ ID NO: 470) DGFFGNPR, (SEQ ID NO: 471)GFPSVLR, (SEQ ID NO: 472) EQLNLR; Chymotrypsin: (SEQ ID NO: 473)TRQNGEAVKTHTNISESHPNATF, (SEQ ID NO: 474) VSCENSPSDTSSVAVGCL,(SEQ ID NO: 475) KNNSDISSTRGFPSVL, (SEQ ID NO: 476) VTSAPMPEPQAPGRY,(SEQ ID NO: 477) TCTVTHTDLPSPL, (SEQ ID NO: 478) SAVGEASICEDDW,(SEQ ID NO: 479) CVPDQDTAIRVF, (SEQ ID NO: 480) KQTISRPKGVAL,(SEQ ID NO: 481) SPRQIQVSW, (SEQ ID NO: 482) TCRVDHRGL, (SEQ ID NO: 483)RESATITCL, (SEQ ID NO: 484) LPPAREQL, (SEQ ID NO: 485) GNPRKSKL,(SEQ ID NO: 486) TVSEEEW, (SEQ ID NO: 487) TIKESDW, (SEQ ID NO: 488)SDTAGTCY, (SEQ ID NO: 489) DSVTISW, (SEQ ID NO: 490) LPSKDVM,(SEQ ID NO: 491) VPPRDGF, (SEQ ID NO: 492) HRPDVY, (SEQ ID NO: 493)QQNASSM, (SEQ ID NO: 494) LPDSITL, (SEQ ID NO: 495) ICQATGF,(SEQ ID NO: 496) NSGERF; Glu-C (phosphate buffer): (SEQ ID NO: 497)LPSPLKQTISRPKGVALHRPD, (SEQ ID NO: 498) HRGLTFQQNASSMCVPD,(SEQ ID NO: 499) KSTGKPTLYNVSLVMSD, (SEQ ID NO: 500) VFVQWMQRGQPLSPE,(SEQ ID NO: 501) SGPTTYKVTSTLTIKE, (SEQ ID NO: 502) HVVCKVQHPNGNKE,(SEQ ID NO: 503) SITLSWKYKNNSD, (SEQ ID NO: 504) SATITCLVTGFSPAD,(SEQ ID NO: 505) LPPKVSVFVPPRD, (SEQ ID NO: 506) WLGQSMFTCRVD,(SEQ ID NO: 507) SVTISWTRQNGE, (SEQ ID NO: 508) SHPNATFSAVGE,(SEQ ID NO: 509) RFTCTVTHTD, (SEQ ID NO: 510) TSSVAVGCLAQD,(SEQ ID NO: 511) AVKTHTNISE, (SEQ ID NO: 512) KNVPLPVIAE,(SEQ ID NO: 513) VYLLPPARE, (SEQ ID NO: 514) GKQVGSGVTTD,(SEQ ID NO: 515) TYTCVAHE, (SEQ ID NO: 516) ALPNRVTE, (SEQ ID NO: 517)VMQGTDE, (SEQ ID NO: 518) QLNLRE; Glu-C (bicarbonate buffer):(SEQ ID NO: 519) SGPTTYKVTSTLTIKE, (SEQ ID NO: 520) GKQVGSGVTTDQVQAE,(SEQ ID NO: 521) HVVCKVQHPNGNKE, (SEQ ID NO: 522) GSASAPTLFPLVSCE,(SEQ ID NO: 523) SHPNATFSAVGE, (SEQ ID NO: 524) AVKTHTNISE,(SEQ ID NO: 525) KNVPLPVIAE, (SEQ ID NO: 526) TYTCVAHE, (SEQ ID NO: 527)ALPNRVTE, (SEQ ID NO: 528) DDWNSGE, (SEQ ID NO: 529) QLNLRE; Asp-N:(SEQ ID NO: 530) DLPSPLKQTISRPKGVALHR P, (SEQ ID NO: 531)DHRGLTFQQNASSMCVP, (SEQ ID NO: 532) DKSTGKPTLYNVSLVMS, (SEQ ID NO: 533)DWNSGERFTCTVTHT, (SEQ ID NO: 534) DSITLSWKYKNNS, (SEQ ID NO: 535)DWLGQSMFTCRV, (SEQ ID NO: 536) DTSSVAVGCLAQ, (SEQ ID NO: 537) DTAGTCY;IgD Peptides from Constant Region Trypsin: (SEQ ID NO: 538)SLWNAGTSVTCTLNHPSLPPQR, (SEQ ID NO: 539) VPAPPSPQPATYTCVVSHEDSR,(SEQ ID NO: 540) DSYYMTSSQLSTPLQQWR, (SEQ ID NO: 541)AQASSVPTAQPQAEGSLAK, (SEQ ID NO: 542) SLEVSYVTDHGPMK, (SEQ ID NO: 543)ATFTCFVVGSDLK, (SEQ ID NO: 544) VPTGGVEEGLLER, (SEQ ID NO: 545)APDVFPIISGCR, (SEQ ID NO: 546) DAHLTWEVAGK, (SEQ ID NO: 547)HSNGSQSQHSR, (SEQ ID NO: 548) CVVQHTASK, (SEQ ID NO: 11) EPAAQAPVK,(SEQ ID NO: 549) TFPEIQR, (SEQ ID NO: 550) ATTAPATTR, (SEQ ID NO: 551)EEQEER, (SEQ ID NO: 552) TLLNASR, (SEQ ID NO: 553) WPESPK; Lys-C:(SEQ ID NO: 554) AQASSVPTAQPQAEGSLAK, (SEQ ID NO: 555)ATTAPATTRNTGRGGEEK, (SEQ ID NO: 556) APDVFPIISGCRHPK, (SEQ ID NO: 557)ATFTCFVVGSDLK, (SEQ ID NO: 558) EIFRWPESPK, (SEQ ID NO: 559)DAHLTWEVAGK, (SEQ ID NO: 560) EEQEERETK, (SEQ ID NO: 561) CVVQHTASK;Arg-C: (SEQ ID NO: 562) QGEYKCVVQHTASKSKKEIFR, (SEQ ID NO: 563)SLWNAGTSVTCTLNHPSLPPQR, (SEQ ID NO: 564) VPAPPSPQPATYTCVVSHED SR,(SEQ ID NO: 565) DSYYMTSSQLSTPLQQWR, (SEQ ID NO: 566) GGEEKKKEKEKEEQEER,(SEQ ID NO: 567) SLEVSYVTDHGPMK, (SEQ ID NO: 568) HSNGSQSQHSR,(SEQ ID NO: 569) TFPEIQR, (SEQ ID NO: 570) TLLNASR; Chymotrypsin:(SEQ ID NO: 571) RWPESPKAQASSVPTAQPQAEGSL, (SEQ ID NO: 572)GTQSQPQRTFPEIQRRDSY, (SEQ ID NO: 573) KCVVQHTASKSKKEIF, (SEQ ID NO: 574)ERHSNGSQSQHSRL, (SEQ ID NO: 575) EVAGKVPTGGVEEGL, (SEQ ID NO: 576)APARPPPQPGSTTF, (SEQ ID NO: 577) RVPAPPSPQPATY, (SEQ ID NO: 578)TCVVSHEDSRTL, (SEQ ID NO: 579) EDQREVNTSGF, (SEQ ID NO: 580)REPAAQAPVKL, (SEQ ID NO: 581) NHPSLPPQRL, (SEQ ID NO: 582) ASSDPPEAASW,(SEQ ID NO: 583) HPTSVTVTW, (SEQ ID NO: 584) NAGTSVTCTL,(SEQ ID NO: 585) VTDHGPM, (SEQ ID NO: 586) TPAVQDL, (SEQ ID NO: 587)RDKATF; Glu-C (phosphate buffer): (SEQ ID NO: 588) SYYMTSSQLSTPLQQWRQGE,(SEQ ID NO: 589) GSLAKATTAPATTRNTGRGGEE, (SEQ ID NO: 590)CPSHTQPLGVYLLTPAVQD, (SEQ ID NO: 591) YKCVVQHTASKSKKE, (SEQ ID NO: 592)VSGFSPPNILLMWLE, (SEQ ID NO: 593) SPKAQASSVPTAQPQAE, (SEQ ID NO: 594)VFPIISGCRHPKD, (SEQ ID NO: 595) SRTLLNASRSLE, (SEQ ID NO: 596)KATFTCFVVGSD, (SEQ ID NO: 597) VAGKVPTGGVEE, (SEQ ID NO: 598) AASWLLCE,(SEQ ID NO: 599) IFRWPE, (SEQ ID NO: 600) AHLTWE;Glu-C (bicarbonate buffer): (SEQ ID NO: 601) GSLAKATTAPATTRNTGRGGEE,(SEQ ID NO: 602) YKCVVQHTASKSKKE, (SEQ ID NO: 603) VSGFSPPNILLMWLE,(SEQ ID NO: 604) SPKAQASSVPTAQPQAE, (SEQ ID NO: 605) DSRTLLNASRSLE,(SEQ ID NO: 606) VSYVTDHGPMK, (SEQ ID NO: 607) VAGKVPTGGVEE,(SEQ ID NO: 608) AASWLLCE; Asp-N: (SEQ ID NO: 609) DSRTLLNASRSLEVSYVT,(SEQ ID NO: 610) DVFPIISGCRHPK, (SEQ ID NO: 611) DKATFTCFVVGS;IgE Peptides from Constant Region Trypsin: (SEQ ID NO: 612)TYTCQVTYQGHTFEDSTK, (SEQ ID NO: 613) GVSAYLSRPSPFDLFIR, (SEQ ID NO: 614)AAPEVYAFATPEWPGSR, (SEQ ID NO: 615) VAHTPSSTDWVDNK, (SEQ ID NO: 616)SPTITCLVVDLAPSK, (SEQ ID NO: 617) NGTLTVTSTLPVGTR, (SEQ ID NO: 618)AVHEAASPSQTVQR, (SEQ ID NO: 619) DWIEGETYQCR, (SEQ ID NO: 620)ASGKPVNHSTR, (SEQ ID NO: 621) GTVNLTWSR, (SEQ ID NO: 12) GSGFFVFSR,(SEQ ID NO: 622) VTHPHLPR, (SEQ ID NO: 623) DFTPPTVK, (SEQ ID NO: 624)HSTTQPR, (SEQ ID NO: 625) HWLSDR, (SEQ ID NO: 626) TFSVCSR,(SEQ ID NO: 627) AEWEQK, (SEQ ID NO: 628) QMFTCR, (SEQ ID NO: 629)DEFICR, (SEQ ID NO: 630) AVSVNPGK, (SEQ ID NO: 631) CADSNPR; Lys-C:(SEQ ID NO: 632) TSGPRAAPEVYAFATPEWPGSRDK, (SEQ ID NO: 633)GSGFFVFSRLEVTRAEWEQK, (SEQ ID NO: 634) QMFTCRVAHTPSSTDWVDNK,(SEQ ID NO: 635) TFSVCSRDFTPPTVK, (SEQ ID NO: 636) SPTITCLVVDLAPSK,(SEQ ID NO: 637) GTVNLTWSRASGK, (SEQ ID NO: 638) PVNHSTRK  Arg-C:(SEQ ID NO: 639) VAHTPSSTDWVDNKTFSVCSR, (SEQ ID NO: 640)GVSAYLSRPSPFDLFIR, (SEQ ID NO: 641) AAPEVYAFATPEWPGSR, (SEQ ID NO: 642)AEWEQKDEFICR, (SEQ ID NO: 643) NGTLTVTSTLPVGTR, (SEQ ID NO: 644)AVHEAASPSQTVQR, (SEQ ID NO: 645) DWIEGETYQCR, (SEQ ID NO: 646)KTKGSGFFVFSR, (SEQ ID NO: 647) ASGKPVNHSTR, (SEQ ID NO: 648) VTHPHLPR,(SEQ ID NO: 649) STTKTSGPR, (SEQ ID NO: 650) HSTTQPR, (SEQ ID NO: 651)KEEKQR, (SEQ ID NO: 652) AVSVNPGK  Chymotrypsin: (SEQ ID NO: 653)ICRAVHEAASPSQTVQRAVSVNPGK, (SEQ ID NO: 654) SRASGKPVNHSTRKEEKQRNGTL,(SEQ ID NO: 655) EDSTKKCADSNPRGVSAY, (SEQ ID NO: 656) RSTTKTSGPRAAPEVY,(SEQ ID NO: 657) TRCCKNIPSNATSVTL, (SEQ ID NO: 658) QSSCDGGGHFPPTIQL,(SEQ ID NO: 659) ATPEWPGSRDKRTL, (SEQ ID NO: 660) QCRVTHPHLPRAL,(SEQ ID NO: 661) TCRVAHTPSSTDW, (SEQ ID NO: 662) TVTSTLPVGTRDW,(SEQ ID NO: 663) IRKSPTITCL, (SEQ ID NO: 664) STASTTQEGEL,(SEQ ID NO: 665) MPEDISVQW, (SEQ ID NO: 666) TPGTINITW, (SEQ ID NO: 667)EVTRAEW, (SEQ ID NO: 668) APSKGTVNL, (SEQ ID NO: 669) TPPTVKIL,(SEQ ID NO: 670) SVCSRDF, (SEQ ID NO: 671) EQKDEF, (SEQ ID NO: 672)ASTQSEL, (SEQ ID NO: 673) VDNKTF; Glu-C (phosphate buffer):(SEQ ID NO: 674) ARHSTTQPRKTKGSGFFVFSRLE, (SEQ ID NO: 675)KQRNGTLTVTSTLPVGTRD, (SEQ ID NO: 676) SNPRGVSAYLSRPSPFD,(SEQ ID NO: 677) RTYTCQVTYQGHTFE, (SEQ ID NO: 678) AASPSQTVQRAVSVNPGK,(SEQ ID NO: 679) LFIRKSPTITCLVVD, (SEQ ID NO: 680) KRTLACLIQNFMPE,(SEQ ID NO: 681) FTPPTVKILQSSCD, (SEQ ID NO: 682) LTLSQKHWLSD,(SEQ ID NO: 683) NKTFSVCSRD, (SEQ ID NO: 684) ISVQWLHNE,(SEQ ID NO: 685) FICRAVHE, (SEQ ID NO: 686) LSTASTTQE, (SEQ ID NO: 687)VYAFATPE, (SEQ ID NO: 688) STKKCAD, (SEQ ID NO: 689) LASTQSE,(SEQ ID NO: 690) WPGSRD; Glu-C (bicarbonate buffer): (SEQ ID NO: 691)KQRNGTLTVTSTLPVGTRDWIE, (SEQ ID NO: 692) WPGSRDKRTLACLIQNFMPE,(SEQ ID NO: 693) AASPSQTVQRAVSVNPGK, (SEQ ID NO: 694) DGQVMDVDLSTASTTQE,(SEQ ID NO: 695) DISVQWLHNE, (SEQ ID NO: 696) FICRAVHE, (SEQ ID NO: 697)VYAFATPE, (SEQ ID NO: 698) LASTQSE; Asp-N: (SEQ ID NO: 699)DRTYTCQVTYQGHTFE, (SEQ ID NO: 700) DSNPRGVSAYLSRPSPF, (SEQ ID NO: 701)DKRTLACLIQNFMPE, (SEQ ID NO: 702) DLFIRKSPTITCLVV, (SEQ ID NO: 703)DISVQWLHNEVQLP, (SEQ ID NO: 704) DFTPPTVKILQSSC, (SEQ ID NO: 705)DNKTFSVCSR, (SEQ ID NO: 706) DSTKKCA; Igκ Peptides from Constant RegionTrypsin: (SEQ ID NO: 707) VDNALQSGNSQESVTEQDSK, (SEQ ID NO: 9)TVAAPSVFIFPPSDEQLK, (SEQ ID NO: 708) VYACEVTHQGLSSPVTK, (SEQ ID NO: 709)SGTASVVCLLNNFYPR, (SEQ ID NO: 710) DSTYSLSSTLTLSK; Lys-C:(SEQ ID NO: 711) VDNALQSGNSQESVTEQDSK, (SEQ ID NO: 712)SGTASVVCLLNNFYPREAK, (SEQ ID NO: 713) TVAAPSVFIFPPSDEQLK,(SEQ ID NO: 714) VYACEVTHQGLSSPVTK, (SEQ ID NO: 715) DSTYSLSSTLTLSK,(SEQ ID NO: 716) SFNRGEC; Chymotrypsin: (SEQ ID NO: 717)QSGNSQESVTEQDSKDSTY, (SEQ ID NO: 718) YPREAKVQW, (SEQ ID NO: 719)IFPPSDEQL, (SEQ ID NO: 720) KSGTASVVCL, (SEQ ID NO: 721) ACEVTHQGL,(SEQ ID NO: 722) SSPVTKSF, (SEQ ID NO: 723) EKHKVY, (SEQ ID NO: 724)TVAAPSVF; Glu-C (phosphate buffer): (SEQ ID NO: 725)QLKSGTASVVCLLNNFYPRE, (SEQ ID NO: 726) VTHQGLSSPVTKSFNRGE,(SEQ ID NO: 727) TVAAPSVFIFPPSDE, (SEQ ID NO: 728) STYSLSSTLTLSKAD,(SEQ ID NO: 729) NALQSGNSQE, (SEQ ID NO: 730) KHKVYACE, (SEQ ID NO: 731)AKVQWKVD; Glu-C (bicarbonate buffer): (SEQ ID NO: 732)QDSKDSTYSLSSTLTLSKADYE, (SEQ ID NO: 733) QLKSGTASVVCLLNNFYPRE,(SEQ ID NO: 734) AKVQWKVDNALQSGNSQE, (SEQ ID NO: 735)VTHQGLSSPVTKSFNRGE, (SEQ ID NO: 736) TVAAPSVFIFPPSDE, (SEQ ID NO: 737)KHKVYACE; Asp-N: (SEQ ID NO: 738) NALQSGNSQESVTEQ, (SEQ ID NO: 739)DSTYSLSSTLTLSKA, (SEQ ID NO: 740) TVAAPSVFIFPPS;Igλ1 Peptides from Constant Region Trypsin: (SEQ ID NO: 741)ATLVCLISDFYPGAVTVAWK, (SEQ ID NO: 742) ANPTVTLFPPSSEELQANK,(SEQ ID NO: 743) YAASSYLSLTPEQWK, (SEQ ID NO: 744) SYSCQVTHEGSTVEK,(SEQ ID NO: 745) AGVETTKPSK, (SEQ ID NO: 746) TVAPTECS; Lys-C:(SEQ ID NO: 747) ATLVCLISDFYPGAVTVAWK, (SEQ ID NO: 748)ANPTVTLFPPSSEELQANK, (SEQ ID NO: 749) SHRSYSCQVTHEGSTVEK,(SEQ ID NO: 750) YAASSYLSLTPEQWK, (SEQ ID NO: 751) TVAPTECS,(SEQ ID NO: 752) AGVETTK, (SEQ ID NO: 753) ADGSPVK; Arg-C:(SEQ ID NO: 754) SYSCQVTHEGSTVEKTVAPTECS; Chymotrypsin: (SEQ ID NO: 755)KADGSPVKAGVETTKPSKQSNNKY, (SEQ ID NO: 756) SCQVTHEGSTVEKTVAPTECS,(SEQ ID NO: 757) GQPKANPTVTL, (SEQ ID NO: 758) YPGAVTVAW,(SEQ ID NO: 759) FPPSSEEL, (SEQ ID NO: 760) KSHRSY, (SEQ ID NO: 761)QANKATL; Glu-C (phosphate buffer): (SEQ ID NO: 762)TTKPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 763) GQPKANPTVTLFPPSSEE,(SEQ ID NO: 764) QWKSHRSYSCQVTHE, (SEQ ID NO: 765) LQANKATLVCLISD,(SEQ ID NO: 766) FYPGAVTVAWKAD, (SEQ ID NO: 904) GSPVKAGVE,(SEQ ID NO: 767) KTVAPTE; Glu-C (bicarbonate buffer): (SEQ ID NO: 768)TTKPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 769) GQPKANPTVTLFPPSSEE,(SEQ ID NO: 770) QWKSHRSYSCQVTHE, (SEQ ID NO: 771) KTVAPTE; Asp-N:(SEQ ID NO: 772) DFYPGAVTVAWKA; Igλ2 Peptides from Constant RegionTrypsin: (SEQ ID NO: 773) ATLVCLISDFYPGAVTVAWK, (SEQ ID NO: 774)AAPSVTLFPPSSEELQANK, (SEQ ID NO: 775) YAASSYLSLTPEQWK, (SEQ ID NO: 776)SYSCQVTHEGSTVEK, (SEQ ID NO: 10) AGVETTTPSK, (SEQ ID NO: 777) TVAPTECS,(SEQ ID NO: 778) ADSSPVK; Lys-C: (SEQ ID NO: 779) ATLVCLISDFYPGAVTVAWK,(SEQ ID NO: 780) SHRSYSCQVTHEGSTVEK, (SEQ ID NO: 781)AAPSVTLFPPSSEELQANK, (SEQ ID NO: 782) YAASSYLSLTPEQWK, (SEQ ID NO: 783)AGVETTTPSK, (SEQ ID NO: 784) TVAPTECS, (SEQ ID NO: 785) ADSSPVK; Arg-C:(SEQ ID NO: 786) SYSCQVTHEGSTVEKTVAPTECS; Chymotrypsin: (SEQ ID NO: 787)KADSSPVKAGVETTTPSKQSNNKY, (SEQ ID NO: 788) SCQVTHEGSTVEKTVAPTECS,(SEQ ID NO: 789) GQPKAAPSVTL, (SEQ ID NO: 790) YPGAVTVAW,(SEQ ID NO: 791) FPPSSEEL, (SEQ ID NO: 792) KSHRSY, (SEQ ID NO: 793)QANKATL; Glu-C (phosphate buffer): (SEQ ID NO: 794)TTTPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 795) QWKSHRSYSCQVTHE,(SEQ ID NO: 796) GQPKAAPSVTLFPPSSEE, (SEQ ID NO: 797) LQANKATLVCLISD,(SEQ ID NO: 798) FYPGAVTVAWKAD, (SEQ ID NO: 799) SSPVKAGVE,(SEQ ID NO: 800) KTVAPTE; Glu-C (bicarbonate buffer): (SEQ ID NO: 801)TTTPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 802) QWKSHRSYSCQVTHE,(SEQ ID NO: 803) GQPKAAPSVTLFPPSSEE, (SEQ ID NO: 804) KTVAPTE; Asp-N:(SEQ ID NO: 805) DFYPGAVTVAWKA; Igλ3 Peptides from Constant RegionTrypsin: (SEQ ID NO: 806) ATLVCLISDFYPGAVTVAWK, (SEQ ID NO: 807)AAPSVTLFPPSSEELQANK, (SEQ ID NO: 808) YAASSYLSLTPEQWK, (SEQ ID NO: 809)SYSCQVTHEGSTVEK, (SEQ ID NO: 810) AGVETTTPSK, (SEQ ID NO: 811) TVAPTECS;Lys-C: (SEQ ID NO: 812) ATLVCLISDFYPGAVTVAWK, (SEQ ID NO: 813)AAPSVTLFPPSSEELQANK, (SEQ ID NO: 814) YAASSYLSLTPEQWK, (SEQ ID NO: 815)SYSCQVTHEGSTVEK, (SEQ ID NO: 816) AGVETTTPSK, (SEQ ID NO: 817) TVAPTECS,(SEQ ID NO: 818) ADSSPAK; Chymotrypsin: (SEQ ID NO: 819)KADSSPAKAGVETTTPSKQSNNKY, (SEQ ID NO: 820) SCQVTHEGSTVEKTVAPTECS,(SEQ ID NO: 821) GQPKAAPSVTL, (SEQ ID NO: 822) YPGAVTVAW,(SEQ ID NO: 823) FPPSSEEL, (SEQ ID NO: 824) KSHKSY, (SEQ ID NO: 825)QANKATL; Glu-C (phosphate buffer): (SEQ ID NO: 826)TTTPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 827) QWKSHKSYSCQVTHE,(SEQ ID NO: 828) GQPKAAPSVTLFPPSSEE, (SEQ ID NO: 829) LQANKATLVCLISD,(SEQ ID NO: 830) FYPGAVTVAWKAD, (SEQ ID NO: 831) SSPAKAGVE,(SEQ ID NO: 832) KTVAPTE; Glu-C (bicarbonate buffer): (SEQ ID NO: 833)TTTPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 834) QWKSHKSYSCQVTHE,(SEQ ID NO: 835) GQPKAAPSVTLFPPSSEE, (SEQ ID NO: 836) KTVAPTE; Asp-N:(SEQ ID NO: 837) DFYPGAVTVAWKA; Igλ6 Peptides from Constant RegionTrypsin: (SEQ ID NO: 838) AAPSVTLFPPSSEELQANK, (SEQ ID NO: 839)YAASSYLSLTPEQWK, (SEQ ID NO: 840) ATLVCLISDFYPGAVK, (SEQ ID NO: 841)ADGSPVNTGVETTTPSK, (SEQ ID NO: 842) SYSCQVTHEGSTVEK, (SEQ ID NO: 843)TVAPAECS; Lys-C: (SEQ ID NO: 844) SHRSYSCQVTHEGSTVEK, (SEQ ID NO: 845)AAPSVTLFPPSSEELQANK, (SEQ ID NO: 846) YAASSYLSLTPEQWK, (SEQ ID NO: 847)ATLVCLISDFYPGAVK, (SEQ ID NO: 848) ADGSPVNTGVETTTPSK, (SEQ ID NO: 849)TVAPAECS; Arg-C: (SEQ ID NO: 850) SYSCQVTHEGSTVEKTVAPAECS; Chymotrypsin:(SEQ ID NO: 851) KADGSPVNTGVETTTPSKQSNNKY, (SEQ ID NO: 852)SCQVTHEGSTVEKTVAPAECS, (SEQ ID NO: 853) GQPKAAPSVTL, (SEQ ID NO: 854)YPGAVKVAW, (SEQ ID NO: 855) FPPSSEEL, (SEQ ID NO: 856) KSHRSY,(SEQ ID NO: 857) QANKATL; Glu-C (phosphate buffer): (SEQ ID NO: 858)TTTPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 859) QWKSHRSYSCQVTHE,(SEQ ID NO: 860) GQPKAAPSVTLFPPSSEE, (SEQ ID NO: 861) LQANKATLVCLISD,(SEQ ID NO: 862) FYPGAVKVAWKAD, (SEQ ID NO: 863) GSPVNTGVE,(SEQ ID NO: 864) KTVAPAE; Glu-C (bicarbonate buffer): (SEQ ID NO: 865)TTTPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 866) QWKSHRSYSCQVTHE,(SEQ ID NO: 867) GQPKAAPSVTLFPPSSEE, (SEQ ID NO: 868) KTVAPAE; Asp-N:(SEQ ID NO: 869) DFYPGAVKVAWKA; Igλ7 Peptides from Constant RegionTrypsin: (SEQ ID NO: 870) ATLVCLVSDFYPGAVTVAWK, (SEQ ID NO: 871)AAPSVTLFPPSSEELQANK, (SEQ ID NO: 872) YAASSYLSLTPEQWK, (SEQ ID NO: 873)VTHEGSTVEK, (SEQ ID NO: 874) VGVETTKPSK, (SEQ ID NO: 875) TVAPAECS;Lys-C: (SEQ ID NO: 876) ATLVCLVSDFYPGAVTVAWK, (SEQ ID NO: 877)SHRSYSCRVTHEGSTVEK, (SEQ ID NO: 878) AAPSVTLFPPSSEELQANK,(SEQ ID NO: 879) YAASSYLSLTPEQWK, (SEQ ID NO: 880) TVAPAECS,(SEQ ID NO: 881) VGVETTK, (SEQ ID NO: 882) ADGSPVK; Arg-C:(SEQ ID NO: 883) VTHEGSTVEKTVAPAECS; Chymotrypsin: (SEQ ID NO: 884)KADGSPVKVGVETTKPSKQSNNKY, (SEQ ID NO: 885) SCRVTHEGSTVEKTVAPAECS,(SEQ ID NO: 886) GQPKAAPSVTL, (SEQ ID NO: 887) YPGAVTVAW,(SEQ ID NO: 888) FPPSSEEL, (SEQ ID NO: 889) KSHRSY, (SEQ ID NO: 890)QANKATL; Glu-C (phosphate buffer): (SEQ ID NO: 891)TTKPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 892) QWKSHRSYSCRVTHE,(SEQ ID NO: 893) GQPKAAPSVTLFPPSSEE, (SEQ ID NO: 894) LQANKATLVCLVSD,(SEQ ID NO: 895) FYPGAVTVAWKAD, (SEQ ID NO: 896) GSPVKVGVE,(SEQ ID NO: 897) KTVAPAE; Glu-C (bicarbonate buffer): (SEQ ID NO: 898)TTKPSKQSNNKYAASSYLSLTPE, (SEQ ID NO: 899) QWKSHRSYSCRVTHE,(SEQ ID NO: 900) GQPKAAPSVTLFPPSSEE, (SEQ ID NO: 901) KTVAPAE; andAsp-N: (SEQ ID NO: 902) DFYPGAVTVAWKA.

DEFINITIONS

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal Thus, the subject can be a human or veterinarypatient. The term “patient” refers to a subject under the treatment of aclinician, e.g., physician.

The term “biological sample” refers to any sample from a subject thatmay contain immunoglobulin secreted from plasma cells. Non-limitingexamples include blood, urine, plasma, and serum.

The terms “peptide,” “protein,” and “polypeptide” are usedinterchangeably to refer to a natural or synthetic molecule comprisingtwo or more amino acids linked by the carboxyl group of one amino acidto the alpha amino group of another.

As used herein, the term “amino acid sequence” refers to a list ofabbreviations, letters, characters or words representing amino acidresidues. The amino acid abbreviations used herein are conventional oneletter codes for the amino acids and are expressed as follows: A,alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid;E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine;I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P,proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine;W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1 Quantification of Peptides from ImmunoglobulinConstant and Variable Regions by Liquid Chromatography-Multiple ReactionMonitoring Mass Spectrometry for Assessment of Multiple Myeloma Patients

Materials and Methods

Chemicals and reagents were acquired from Sigma-Aldrich (Milwaukee,Wis.); HPLC solvents were purchased from Burdick and Jackson (Honeywell,Muskegon, Mich.). Standard peptides were synthesized, HPLC-purified,characterized with MALDI MS, QqQ MS, and amino acid analysis, aspreviously described (Remily-Wood E R, et al Proteomics Clin Appl 2011,5, 383-96).

Sample Collection and Summary of Patient Data

De-identified serum was collected from patients in accordance withprotocols approved by the University of South Florida InstitutionalReview Board. Blood was collected in serum separator tubes (BD, FranklinLakes, N.J.), clotted for 30 minutes, spun down at 3,600 rpm for 10minutes (5702, Eppendorf), and refrigerated until analysis (t<3 weeks).Samples (n=83) were collected. The study population contained 46 malesand 37 females between ages 34 and 87 (median age 63) with diagnosesincluding MM (45), smoldering MM (3), light chain only MM (1),non-secretory MM (1) MGUS (6), plasmacytoma (5), plasma cell leukemia(2), Waldenstrom's macroglobinemia (8), Non-Hodgkin's Lymphoma (6),other leukemias or lymphomas (4), amyloidosis (2), and prostate cancer(1). Samples were selected to represent all types of PCDs with varyinglevels of immunoglobulin expression and age-matched patients with otherdiseases. Of these patients, 71 had elevated levels of immunoglobulinexpression detected by SPEP compared to reference values for healthycontrols. Twelve patients had immunoglobulin expression levelscomparable to healthy controls and were not detectable by SPEP (SPEP−).

Bone marrow aspirates were collected from 2 patients (not included inthe cohort used for the constant region analysis). MM tumor cells wereobtained by Ficoll and CD138⁺ plasma cell selection (Miltenyi, Auburn,Calif.). An aliquot of 0.5 million cells was used for RNA-sequencing.

Clinical Measurements

SPEP was performed using capillary zone electrophoresis (Capillarys,Sebia). Immunoglobulin concentrations were calculated using measurementsof total serum protein (Fusion 5.1FS Chemistry Analyzer, Ortho ClinicalDiagnostics, NJ). Immunotyping was performed using monospecific antiserafor IgG, IgA, and IgM heavy chains as well as κ and λ light chains(Capillarys, Sebia) Immunofixation electrophoresis (SPIFE 3000, HelenaLaboratories) was used to confirm serum immunotyping results or to testfor IgD and IgE. Nephelometry of the immunoglobulins was performed(Vitros 5.1 FS Chemistry System, Ortho-Clinical Diagnostics) todetermine the concentration of IgA, IgG, and IgM using goat antisera asthe primary active reagents.

LC-MRM Quantification of Proteins in Serum

Peptide targets were selected from LC-MS/MS data (see FIG. 51). Serumproteins were denatured with 8M urea, reduced with tris(2-carboxyethyl)phosphine, and alkylated with iodoacetamide prior to a ten-fold dilutionin aqueous 30 mM ammonium bicarbonate and in-solution tryptic digestion(Promega, Madison, Wis.). Internal standards were spiked into eachsample (Table 1) (Berth M, et al Clin Chem 1999, 45, 309-10; French MAH,et al Clin Exp Immunol 1984, 56, 473-5; Haraldsson A, et al Ann ClinBiochem 1991, 28, 461-66; Chen K, et al Nephrology 2005, 10, 594-596;Buckley R, et al J Clin Invest 1975, 55, 157-65; Hunder G, et alArthritis Rheum 1974, 17, 955-63). The equivalent of 0.5 nanoliters oftryptically digested serum was injected for each LC-MRM analysis.

TABLE 1 Endogenous Peptides and Labeled Standards used for LC-MRMQuantification of Immunoglobulins. Average Expression & Median MedianReference Intra- Inter- Range Assay Assay Protein (mg/ml) Peptide ISTransitions CV (%) CV (%) Outliers IgG1 5.91 GPSVFPLAPSSK 10.2 y₈-y₁₀7.5 7.2 3 3.19-10.2 (SEQ ID NO: 1) IgG2 3.04 GLPAPIEK 6.63 y₄-y₆ 6.0 5.92 1.23-6.63 (SEQ ID NO: 2) IgG3 0.61 WYVDGVEVHNAK 1.94 y₆, y₈, y₉ 1618.3 12 0.16-1.94 (SEQ ID NO: 3) IgG4 0.24 TTPPVLDSDGSFFLYSR NSy₈, y₁₀, y₁₂ — — — 0.03-1.33 (SEQ ID NO: 4) IgA1 1.88 TPLTATLSK NS y₅-y₇— — — 1.36-2.5  (SEQ ID NO: 5) IgA2 0.54 DASGATFTWTPSSGK* 0.6  y₇-y₁₀ 2116 5 0.28-0.61 (SEQ ID NO: 6) IgA1-2 2.42 WLQGSQELPR 3.1 y₆-y₈ 6.8 9.6 21.64-3.11 (SEQ ID NO: 7) IgM 0.70 DGFFGNPR 2.3 y₄-y₆ 11 7.6 1 0.4-2.3(SEQ ID NO: 8) κ LC 2.31 TVAAPSVFIFPPSDEQLK* 3.0 y₈, y₉, y₁₁ 7.3 6.6 61.55-3.08 (SEQ ID NO: 9) λ LC 1.54 AGVETTTPSK 2.24 y₅-y₇ 19 9.0 160.83-2.24 (SEQ ID NO: 10) IgD 0.0139 EPAAQAPVK 0.50 y₅-y₇ 0.52 — 00.001-0.024 (SEQ ID NO: 11) IgE 0.0001 GSGFFVFSR* 0.50 y₅-y₇ 6.7 — 0    0-0.002 (SEQ ID NO: 12) Albumin 35 LVNEVTEFAK* 3.5 y₅, y₇, y₈ 5.06.3 0 30-40 (SEQ ID NO: 13) Peptides representative for individualproteins and groups of isoforms are quantified. Standard peptides weresynthesized with either isotope-labeled amino acids or with a structuralanalog created by single conservative amino acid replacement (as notedby *) that differs only by a methylene group in the side chain of theunderlined residue. The limit of detection based on internal standardsis also shown along with other monitored peptides that do not havequantitative assays developed. Eleven additional peptides are monitoredto improve confidence in quantification by evaluation of consistency,but SIS peptides have not been synthesized. Reference ranges provided byARUP Laboratories.

LC-MRM was performed using a nanoLC interfaced with a triple quadrupolemass spectrometer (EasyNanoLC and TSQ Quantum Ultra or Vantage, Thermo,San Jose, Calif.), as previously described (see supplement foradditional details) (Remily-Wood E R, et al Proteomics Clin Appl 2011,5, 383-96). Briefly, peptides were desalted on a reversed phasepre-column prior to reversed phase chromatography using 10 minutegradients (C18 Pepmap100, Thermo, San Jose, Calif.). Peptide precursorswere selected with 0.4 Q1 resolution; fragment ions were then selectedwith 0.7 Q3 resolution. Scan width was 0.002, and transitions wereacquired for 5-20 milliseconds. If the peptides were not optimized bymanual infusion, collision energy values were calculated using Equation1 based on the mass-to-charge ratio (m/z) of the doubly protonatedpeptide precursor (MacLean B, et al Bioinformatics, 2010, 26, 966-8;Zhang G, et al J Proteome Res 2011, 10, 305-19).

CE=(0.034*m/z)+3.314 V  Equation 1:

Batches of ten samples were analyzed on the instrument with a reversecalibration curve of the following three standards: a common serumstandard was spiked with stable isotope-labeled standard peptides at theaverage normal abundance, maximum normal abundance, and maximum normalabundance plus 1 mg/ml. Samples were analyzed one through ten, followedby standard samples, and then this batch was repeated for triplicatemeasurements.

Data Analysis

Peak areas (PA) were calculated using MRMer implemented in GenePattern.Raw data files were converted into mz×ml; LC-MRM peaks were extractedand visualized for transition evaluation. Resulting data were assessedfor quality control and compared between patient groups usingPost-MRMer. After data review, protein concentrations (in mg/ml) werecalculated using the PA ratio of the endogenous peptide to itscorresponding standard. The resulting data were evaluated using existingreference ranges for protein-based measurements and compared withnephelometry measurements. Intra-assay CV values were determined usingtriplicate LC-MRM analysis; inter-assay CV values were calculated fromten LC-MRM analyses of different preparations of the same serum samples(n=3). Median values are reported. Batch-to-batch variation was alsoexamined.

Statistical Analyses

To evaluate assay robustness, the data set was filtered for outliers,when CV>0.5 and at least one value calculated to be above the previouslydefined range of healthy controls. Samples below the defined normalmaximum value were not examined for outliers because the low proteinexpression level often contributed to increased CV values. In the caseswith high CV values, an individual measurement was discarded when itsdistance from the median was twice (or greater) the distance of theother data point from the median. Out of 249 data points for eachpeptide, zero to fifteen outliers were removed (see Table 1). Batcheffects were also evaluated using the reverse calibration curves andvisual inspection of the entire dataset for all 83 patient specimens.From the remaining sample points, the mean, median, and standarddeviation were calculated and a two-sided Wilcoxon rank-sum test wasused to determine if a statistical difference existed between theexpression of each protein in patients diagnosed with each specific typeof PCD (as determined by clinical diagnoses for the differentimmunoglobulins or LC-MRM analysis for specific isoforms of IgA and IgG)and all other patient samples. The Holm-Bonferroni method was used toadjust for multiple hypotheses testing with type I error (α), which wasset to 0.05.

RNA Sequencing and LC-MRM Detection of Variable Region Peptides

H929 multiple myeloma cells and two patient specimens (all n=0.5×10⁶cells) were selected for a proof-of-concept experiment in immunoglobulinvariable region sequencing and detection of variable region peptides.mRNA sequencing (RNA-seq) was performed from 100 ng of total RNA usingthe Encore Complete Library System (NuGEN, San Carlos, Calif.).Strand-specific cDNA generated from this kit was used to prepare abarcoded library appropriate for multiplexed massively parallelsequencing. Paired-end 100 base reads (n˜2×10⁷) were generated using theHiScan SQ sequencer (Illumina, San Diego, Calif.). Demultiplexing anddata quality evaluation were performed using CASAVA 1.8.2 (Illumina)RNA-seq reads were aligned to the human hs37d5 reference genome and theGencode v14 gene model using Tophat2. The expressed transcripts wereassembled and evaluated using the de novo assembly software, Trinity.Contiguous sequences are aligned back to the human genome with BLAST,and best hits are manually examined. NCBI ORF identifies potentialprotein constructs. After generating the protein sequence for theimmunoglobulin secreted by the tumor cells, LC-MRM was used to screenfor the detection of tryptic peptides from the Igκ constant and variableregions in H929 conditioned culture media, as described above. Forpatient specimens, both samples were analyzed for LC-MRM detection ofvariable region peptides from Patient 1 to examine their detection inlow levels of disease and against a control background.

Results and Discussion

LC-MRM Quantification of Heavy Chains Using Constant Region Peptides

Triplicate LC-MRM analysis was performed on 83 patient samples. For moststandards, spike-in concentrations equivalent to the maximum value forexpression in healthy controls to enabled rapid patient evaluation(higher endogenous peptide signal indicates disease burden). Spikedamounts of the internal standards for albumin and those common tomultiple IgG isoforms were decreased due to their high abundance inserum. Due to the low abundance of IgD and IgE in healthy controls,their internal standards were spiked at 0.5 mg/ml (or 0.05 g/dl), whichis well below the typical limit for starting treatment after diseaserelapse and half the limit of detection for SPEP. With the exception ofpeptides monitoring IgA isoforms, median CV values were below 20%.

Comparison of SPEP/IFE results to LC-MRM is useful for verification thatthe new technique is able to accurately discern involved immunoglobulinsfor PCD patients. As expected, LC-MRM detection of increased expressionof immunoglobulins matched the clinical diagnoses made by serialSPEP/IFE measurements in all cases when the involved immunoglobulinabundance exceeded the reference range for healthy controls. Sampleswere taken from 19 IgG patients (average SPEP 14.9 mg/ml), 20 IgApatients (average SPEP 14.0 mg/ml), and 17 patients with elevated IgM(average SPEP 10.8 mg/ml). LC-MRM was able to identify increasedexpression in 18/19 patients with IgG disease. For SPEP+ patients withIgA disease, 18/20 could be detected by LC-MRM of the peptide,WLQGSQELPR (SEQ ID NO:7). The relative quantification of the secondpeptide from IgA1 detected elevated expression in one of these patients,but the total IgA expression was in the normal range for the otherpatient. When SPEP detects low levels the monoclonal immunoglobulin butthe total immunoglobulin expression level was still in the referencerange for healthy controls, both LC-MRM and nephelometry were unable todetect the disease. Longitudinal monitoring of these IgG and IgApatients may still indicate the presence of disease and increases intumor burden. All 17 SPEP+ IgM cases could be detected. All IgD (n=5)and IgE (n=1) patients were also correctly identified. Seven patientsdiagnosed with free light chain disease expressed no elevatedconcentrations of any heavy chain immunoglobulin. LC-MRM measurements ofimmunoglobulins were in the normal range for all SPEP− samples. Samplesfrom two patients with biclonal MM diagnoses made by SPEP/IFE wereanalyzed with LC-MRM (one IgG/IgM and one IgA/IgG). Both immunoglobulinchains were detected above the normal range for the IgA/IgG biclonalpatient, but only the elevated IgM expression was detected in theIgM/IgG biclonal patient.

The LC-MRM heavy chain measurements correlated well to the valuesdetermined by nephelometry, the current clinical standard forquantification. Comparison of IgG measurements using the two methods isshown in FIG. 1A. LC-MRM IgG data was compiled by summing the IgG1,IgG2, and IgG3 values. Because the IgG4 could not be quantifiedabsolutely (due to poor synthesis of the SIS peptide), that value isexcluded, and LC-MRM measurements should be slightly less than thosefrom nephelometry (i.e. slope <1). This calculation may also limit thecorrelation (R²=0.82). Correlation was poorer in patients with high IgGexpression levels, perhaps due to LC-MRM saturation from nanoLC columnloading limits. Removal of the samples with saturated IgG detectionincreased the correlation between LC-MRM and nephelometry (R²=0.98). IgAand IgM values were also highly correlated (FIG. 1B and FIG. 1C). Inaddition, both LC-MRM and nephelometry measurements were compared toSPEP data. As expected, LC-MRM and nephelometry correlate better witheach other than with SPEP, due to the fact that they both measure thetotal immunoglobulin rather than the disease-specific M-protein.

LC-MRM has better sensitivity than nephelometry, but has slightly poorerprecision. LC-MRM was able to quantify immunoglobulin concentrations inpatients when nephelometry reported that the values were below thestated limits of quantification: 2.71 mg/ml IgG, 0.41 mg/ml IgA, and0.26 mg/ml IgM. Data are shown for IgG (n=11) in FIG. 1D, IgA (n=29) inFIG. 1E, and IgM (n=34) in FIG. 1F. This improvement in sensitivity mayenable better evaluation of immune paresis, reduction of the populationof other plasma cells due to the clonal expansion of the tumor cells,which could have a bearing on patient prognosis. Intra-assay andinter-assay CV values are approximately 5% and 10% for nephelometry(Alexander Jr R L Clin Chem 1980, 26, 314-7; Guiguet M, et al J ClinChem Biochem 1983, 21, 217-21); LC-MRM intra-assay CV values arecompetitive, but inter-assay CV values are ˜2-fold higher due to theadditional processing steps (Table 1).

LC-MRM of constant region peptides also enables the quantification ofindividual immunoglobulin heavy chain isoforms, which builds on existingclinical methods. This additional information could enable moresensitive detection of disease for patients with MM tumors that secretelower abundance isoforms (e.g. IgG4). In order to assess the value ofthis additional level of detail provided by LC-MRM, the expressionlevels of each immunoglobulin were compared between the patientsexpressing each monoclonal immunoglobulin and those with other types ofPCDs. Box plots are used for visualization (FIG. 2), and statisticalresults are included in Table 2. In FIG. 2A, the IgG patients arecompared to other samples (as described above). IgA patients weredifferentiated using a peptide representing total IgA, WLQGSQELPR (SEQID NO:7) (FIG. 2B). IgM levels were detected using the peptide, DGFFGNPR(SEQ ID NO:8) (FIG. 2C). Box plots are not shown for IgD or IgE, becauseof the low sample size of those patients and limited detection of thoseendogenous peptides. As expected, statistical significance improves asthe normal range of protein expression decreases; in other words,quantification of total immunoglobulin levels is more effective atdetecting disease when the background levels of that immunoglobulin arelower. Therefore, LC-MRM detection of isoforms can further separate IgGpatients (defined by serial SPEP/IFE measurements) into IgG1 (n=11),IgG2 (n=2), IgG3 (n=4), and IgG4 (n=1) based on the elevation ofisoform-specific peptides. Two examples are shown in box plots in FIG.2; statistical results are included in Table 2 to indicate the increasedseparation of the groups and subsequently better detection of elevatedimmunoglobulin levels for IgG isoforms with lower total expression.Patients identified by LC-MRM with IgG1 disease (n=11) had significantlyhigher IgG1 expression levels than other patients (FIG. 2D). Patientswith IgG3 disease (n=4) had significantly higher levels of that isoformthan other patients (FIG. 2E). Due to insufficient sample size, data arenot plotted for IgG2 and IgG4. IgA patients could be separated byisoforms into IgA1 and IgA2, but no IgA2 patients were detected in thiscohort.

TABLE 2 Statistical Evaluation of LC-MRM Performance in SeparatingPatient Groups. Monitored Corresponding Ig Diagnosis Other DiagnosesStatistical Results Protein(s) n Mean SD Median n Mean SD Median z-valuep-value IgA1, 2 21 14.83 16.53 6.20 62 0.66 0.61 0.46 5.10 3.5E⁻⁷ IgA221 0.03 0.04 0.01 62 0.08 0.18 0.03 −1.36 0.17 IgG1, 2, 3 21 16.59 18.5712.40 62 4.26 3.06 3.78 4.92 8.8E⁻⁷ IgG1 10 15.19 5.38 13.74 73 2.401.91 1.81 5.09 3.7E⁻⁷ IgG3 4 30.95 41.11 17.62 79 0.66 0.60 0.53 2.240.02 IgM 18 7.26 9.05 3.69 65 0.10 0.10 0.06 6.45  1.4E⁻¹⁰ κ KLC 3611.99 11.09 8.83 47 2.37 1.72 2.04 6.16  7.2E⁻¹⁰ λ LC 36 12.00 30.001.75 47 0.44 0.63 0.22 5.66 1.6E⁻⁸ IgD 5 6.57 8.37 2.30 78 0.00 0.030.00 7.23  4.8E⁻¹³ Each immunoglobulin measurement is compared betweenthe population of SPEP positive patients and patients with otherdiagnoses. For isoform-specific measurements, the diagnosis of theinvolved immunoglobulin is achieved solely by LC-MRM. In each case, thenumber of patients as well as the mean, standard deviation, and themedian of the protein expression are listed. Statistical significancewas assessed by the Holm-Bonferroni method; z-values and p-values arelisted.

This additional information has the potential to improve patientmonitoring, especially in cases of IgG MM where some isoforms arenaturally high in abundance (such as IgG1 and IgG2) and others aresignificantly lower (IgG3 and IgG4). The patient's total IgGconcentration may fall within the reference range for healthy controls(4.5-20 mg/ml), but a single isoform can be detected by LC-MRM and shownto be significantly overexpressed. For example, the total IgG wasmeasured to be within normal limits for three patients, but elevatedlevels of IgG3 were detected with LC-MRM. This additional capabilityenables more sensitive detection of disease, and it also may implicateinvolvement of multiple immunoglobulins, which could indicate anotherclonal tumor population. As an example, 3 patients were found by LC-MRMquantification to have elevated IgG3 levels that were previouslyundetected (two in IgM patients and one in a patient not presenting withMM).

LC-MRM Quantification of Light Chains using Constant Region Peptides

In addition to monitoring heavy chains and their isoforms, LC-MRM wasused to measure constant regions peptides as surrogates for theexpression of the light chains, κ and λ, and to calculate a κ:λ ratio.Assays were developed for κ: TVAAPSVFIFPPSDEQLK (SEQ ID NO:9) and λ:AGVETTTPSK (SEQ ID NO:10). These measurements could be used to detectdysregulation of light chain expression not only as overexpression butalso by the κ:λ ratio. LC-MRM data was not compared to the results ofSFLC assays, because the values are expected to differ (due to thecomparison of total light chain by LC-MRM to free light chain by SFLC).However, LC-MRM was effective in detecting light chain-only disease inall cases (4/4). Patients with light chain-only disease that could notbe monitored by SFLC were not included in this study, so it is unclearif LC-MRM would have utility there.

To evaluate the separation of patients with κ and λ diagnoses by LC-MRM,box plots are shown in FIG. 3A and FIG. 3B, respectively. Statisticalresults are listed in Table 2. Protein expression levels in patientsdiagnosed with elevated κ were significantly higher than in otherpatients; λ levels were also significantly different in λ patients whencompared to other patients. The κ:λ ratio values have been plotted inlog-scale (FIG. 3C). Data from non-MM patients and those expressingbiclonal light chains (i.e. both κ and λ) were not included, nor weresamples (n=5) where only one light chain could be quantified. Out of the30 K samples, only one patient was found to have a lower than expectedκ:λ ratio (the reference range for total κ:λ in healthy controls is1.3-2.7) (Katzmann J A, et al Clin Chem 2002, 48, 1437-44), and onepatient had a ratio within the normal range. Out of the 30λ patients,two samples were found to have κ:λ ratios above the normal range, andtwo samples fell within the normal limits. Additional discussion on thecharacteristics of these patients is included in the supplement.

Longitudinal Patient Monitoring

An IgE MM patient was monitored using LC-MRM for comparison withexisting protein-based methods (FIG. 4); total IgE quantification ismore sensitive for disease detection than SPEP due to the low levels ofbackground IgE expression (at μg/ml levels). Serum samples were acquiredat diagnosis and each time the patient received treatment. Initially,LC-MRM and SPEP show a distinct M-protein correlating to IgE myeloma,assigned by IFE to confirm the SPEP diagnosis and by endogenous IgEpeptide intensity in LC-MRM (FIG. 4A). However, analyses during thecourse of treatment illustrate the similarity of nephelometry and LC-MRMfor monitoring IgE. SPEP results show no elevated IgE protein in theserum after the fourth and fifth cycles of treatment at 15 and 19 weeksafter diagnosis (FIGS. 4B and 4C); nephelometry and LC-MRM were stillable to detect elevated IgE levels at those time points (FIG. 4D).Compared with SPEP, nephelometry and LC-MRM can better characterize thedisease in this IgE patient, demonstrating another decrease in tumorburden after the fifth cycle of treatment. After the sixth cycle oftreatment and during the subsequent follow-up, neither method coulddetect IgE. Based on the clinical evaluation (and not LC-MRM data),treatment was discontinued after six cycles, and the patient has been inremission.

Detection of Variable Region Peptides for M-Protein Quantification

LC-MRM of constant region peptides has similar performance for detectionof disease with similar intra-assay variability but higher inter-assayvariability as compared with current immunoglobulin quantificationtechniques. One way to improve the LC-MRM method is to define thesequence of the variable region, which could lead to detection of thespecific monoclonal immunoglobulin, similar to SPEP. De novo assemblywas performed from RNA-seq data to determine the Ig sequence secreted bythe H929 cell line. Although NCBI ORF Finder identified severalpotential constructs, a single protein sequence had the expected sizeand the expected constant region primary amino acid sequence. This aminoacid sequence matched that expected from a translation of the IGKC,IGKJ1, and IGKV3-15 regions. Differences at the junctions were detectedas can be expected during Ig recombination: proline was inserted betweenIGKV3-15 and IGKJ1, and arginine was inserted between IGKJ1 and IGKC.For the Igκ secreted by H929 cells, three constant region peptides (C)and 6 variable region peptides (V) could be detected by LC-MRM (FIG. 5).The peptides are: C1: VDNALQSGNSQESVTEQDSK (SEQ ID NO:14), C2:DSTYSLSSTLTLSK (SEQ ID NO:15), C3: TVAAPSVFIFPPSDEQLK (SEQ ID NO:16),V1: ATGIPAR (SEQ ID NO:17), V2: VTLSCR (SEQ ID NO:18), V3: LSVSPGER (SEQID NO:19), V4: LL(ox)MYDASTR (SEQ ID NO:20), V5: ASQSVSSHLVWYQQKPGQAPR(SEQ ID NO:21), and V6: LLMYDASTR (SEQ ID NO:22); the identity of eachpeptide was verified by comparison of RT and fragmentation with LC-MS/MSand standards. Because the specific amino acid sequences of the variableregion peptides from monoclonal immunoglobulin may be contained in otherantibodies secreted by different cells, the quantification of thedisease-specific VRPs are limited by the amount of the peptide producedfrom the background of immunoglobulins secreted by normal plasma cells.Detection of the IgK immunoglobulin from H929 cells by LC-MRM of theLSVSPGER (SEQ ID NO:19) sequence in a background of serum from a healthycontrol could be accomplished over more than two orders of magnitude anddown to a spiked level of 0.6 fmol/μl of serum, which corresponds todetection above background for 0.0015 g/dl. This value represents˜67-fold improvement over the SPEP detection limit at 0.1 g/dl.

In addition, RNA-sequencing and in silico translation also provided theprotein sequences of two K immunoglobulins from patients. Both patientsare currently in remission, but serum samples were collected andanalyzed with LC-MRM for variable region peptides. The first patient wasIgA/K with a current M-protein measurement by SPEP of 0.2 g/dl, which isbelow the 0.5-1 g/dl level used to initiate treatment for relapse; thesecond patient has IgG/K disease with no detectable M-protein at thetime the sample was taken. Therefore, three peptides from the κ lightchain of Patient 1 were tested in both patients to see the specificityof assays for the variable region peptides (FIG. 6). In all cases, thelevel of each variable region peptide was at least 1000-fold higher inPatient 1 than in Patient 2. Based on these preliminary data, thisapproach could generate personalized assays for detection of tumorburden.

CONCLUSIONS

Using reference ranges for current protein-based assays, LC-MRMquantification of immunoglobulins was compared against current clinicalmethods using a sample size of 83 patients. While the LC-MRM assay isslower to run (overnight versus a few minutes), the therapeutic windowfor these patients is long, so treatment decisions do not have to bemade immediately and the additional time required for this assay is notdetrimental to the patients (Koomen J M, et al Mol Cell Proteomics 2008,7, 1780-94). The ability of LC-MRM to monitor all immunoglobulins (andalbumin as well as other proteins) allows for an increase in efficiency,with MM immunoglobulin and isoform quantification accomplished with oneexperiment (and one instrument). The LC-MRM method may improve analysisof the rarer MM types (IgD, IgE, and light chain only disease) thatcurrently require multiple tests for monitoring. Peptide-based LC-MRMquantification of total immunoglobulin expression levels offers slightimprovements in sensitivity over nephelometry, as well as the ability toquantify isoforms, with a trade-off in precision (˜2-fold higher CVvalues). For higher abundance immunoglobins (e.g. IgG1), the precisionis the critical variable for detection of disease because of thebackground level of protein expression, which is a disadvantage forLC-MRM. For lower abundance isoforms and other immunoglobulins (e.g. IgDor IgE), an increase in sensitivity would be critical to detect lowlevels of tumor burden during treatment and relapse. The increase insensitivity can also be used to monitor the amount of immune paresis(reduction in other immunoglobulin levels due to reduction of normalplasma cells from the bone marrow by the growth of the tumor), but thisparameter is not currently factored into patient treatment decisions.

In this experiment, outliers in triplicate measurements were detected atlevels of 6.4% or less; some peptides did not have any outliersdetected. Reduction of CV values and elimination of outliers will becritical for clinical implementation without triplicate analysis.Automation of sample preparation may provide improvements in precision,and injection of larger sample amounts for analytical scalechromatography would be expected to improve on the nanoLC analysis hereby eliminating saturation effects, improving precision, and eliminatingoutliers. If technology shifts to mass spectrometry-based methods, thisset of assays could be clinically utilized. In order to pursue itsimplementation, a large cohort of healthy controls would need to beanalyzed to define the appropriate reference ranges for peptide-basedLC-MRM assays. Furthermore, additional data should be collected forlongitudinal patient monitoring to define the levels of naturalvariation in immunoglobulin abundance in serum and to determine thethreshold for increases in immunoglobulin level that should triggeradditional treatment.

Finally, the determination of variable region sequences and theresulting tryptic peptides is a promising method for improving patientmonitoring with quantitative mass spectrometry. Although this requiresan initial RNA-seq experiment to identify the highly expressedrearrangements, these transcripts can be identified with as few as 20million RNA-seq reads due to the high expression of Ig in multiplemyeloma tumor cells. This method allows for a highly specific trackingof the disease-specific monoclonal protein. This personalized approachcould offer a better understanding of the unique aspects of anindividual patient's disease, but the effectiveness of the assay wouldvary between patients because of the sequence-specific performance ofthe variable region peptides and the patient-specific background ofother immunoglobulins with the same sequence.

An assay platform that measures both constant region peptides for allimmunoglobulins and variable region peptides for the patient'sdisease-specific immunoglobulin could significantly improve onsensitivity. The detection of the disease-specific biomarker is thenpaired with the ability to detect changes in the other immunoglobulins,which could indicate immune paresis (describing disease severity) orexpansion of another MM clone that secretes a different immunoglobulin(i.e. relapse with a different MM tumor). This platform would be of usein monitoring depth of response to therapy, which has prognostic value,and determining disease progression either from premalignant conditions(e.g. MGUS) or in relapse after treatment. Improvements in the abilityto quantify the tumor burden are likely to shape novel clinicalapproaches to MM treatment.

Example 2 Assay Development Using LC-MS/MS of Serum at the Time ofDiagnosis and De Novo Peptide Sequencing to Determine Variable RegionPeptides

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptidesequencing can be used for the detection and identification of variableregion peptides (VRPs) in serum samples or enriched Ig fractions. Unlikethe sequencing of antibodies with affinity capture using a knownantigen, separations methods are used to create fractions of total Ig,and the expression level of the monoclonal Ig specific to disease willneed to be sufficiently high to make the VRPs obvious.

Sample Selection for De Novo Peptide Sequencing

In order to test the potential of LC-MS/MS to define VRPs for assaydevelopment, serum from 10 of the 30 patients in Example 1 with varyingexpression levels (1 mg/ml to >20 mg/ml) are blinded and used fortesting the effectiveness of VRP detection by LC-MS/MS.

De Novo Immunoglobulin Peptide Sequencing

Because of the wide range of expression levels different methods foridentifying variable region peptides from the monoclonal Ig can beemployed. When the expression is very high (e.g. >20 mg/ml or 25% oftotal blood protein), tryptic digests of total serum proteins can becompared to isolate unique peptides in each sample. However, when theexpression is lower, the monoclonal Ig will need to be enriched. Forthat purpose, gel-based SPEP is used with excision of the γ region orSDS-PAGE with excision of heavy (50 kDa) and light chain (25 kDa) bandsprior to LC-MS/MS. For IgG, enrichment with immobilized protein A/G(Ultralink, Pierce) is used for enrichment. All samples are denatured (8M urea), reduced, alkylated, and digested with trypsin. LC-MS/MS isperformed with UPLC (RSLC, Dionex, Sunnyvale, Calif.) and high massaccuracy MS and MS/MS in an hybrid linear ion trap-orbital ion trap massspectrometer (LTQ Orbitrap, Thermo, San Jose, Calif.), as describedpreviously. Peptide sequences are identified by database searches withSequest (Thermo, San Jose, Calif.) and Mascot against the human entriesin the UniProt database containing both forward and reverse proteinsequences to enable assessment of false discovery rate (% reverse out ofthe total). Scaffold will assemble data to define and eliminate thecommon background.

For the samples with the highest abundance Ig expression, SIEVE is usedto compare replicates of one sample against all others to find uniquepeptides, which are manually sequenced de novo using high resolution andaccurate mass MS and MS/MS data. In addition, ScanRanker is used forsequence tagging, identifying peptides using the MS/MS data first.Comparisons of these results between samples indicates the uniquepeptides in each sample. Finally, conserved amino acids and sequences inthe variable region of the immunoglobulins are used to search forpatterns in the MS/MS data or for peptides with specific cleavage sites(and therefore specific C-termini) These strategies are applied to rawserum digests and enriched Ig fractions to define the amount of Igexpression required for each method to be effective. VRPs detected byLC-MS/MS are compared to the RNA-seq to determine the ability to developthese assays from serum without the need for bone marrow.

Example 3 Determination of the Sequence of the Immunoglobulin (Ig)Secreted by the Multiple Myeloma Tumor Cells Using RNA-Sequencing(RNA-Seq) Using CD138+ Cells Selected from Bone Marrow Aspirates

The example data involved 100 base read lengths (e.g. HiSEq, Illumina),but longer read lengths produced by other instruments (e.g. 300 basereads with MiSeq, Illumina) would be advantageous for data quality.Attempts are made to reconstruct the heavy chain constant regions(IgG1-4, IgA1-2, IgM, IgD, and IgE), the light chain constant regions(Igκ and Igλ), the joining regions, and the variable regions for eachimmunoglobulin. Successful reads should assemble constant region,joining region, and variable region for the intact immunoglobulin. Bothheavy chain and light chain can be important for patient monitoring.

mRNA enrichment can be used to improve the application of sequencing andreduce the total amount of sequencing required for each patient.

5′ RACE can also be used for targeted sequencing to increase the numberof reads for the immunoglobulins and decrease the overall need forsequencing per patient. Briefly, since the constant regions of theantibody are at the C-terminus, primers to the beginning of theC-terminus of the constant region can be used to read back through thejoining region and the variable region. Primers can be designed againstthe heavy chains and their isoforms as well as the light chains andtheir isoforms.

Data from the above methods are assembled and translated to protein. Theprotein sequence is used to define peptide biomarkers that can bemeasured with quantitative mass spectrometry. The Ig sequence is blastedagainst existing publically available databases and previous patients todefine the most unique parts of the sequence.

The Ig sequence is digested in silico to generate peptides that containthe maximally unique sequence. Multiple enzymes are tested, includingbut not limited to trypsin, endoproteinase Lys-C, chymotrypsin,endoproteinase Glu-C, Asp-N, Arg-C, etc. The effectiveness of thepeptide lies in its uniqueness and its ability to be detected by themass spectrometer.

Heavy and light chain proteins are isolated by SDS-PAGE and excised forin-gel digestion. Alternatively, the filter-aided sample preparation(FASP) can be used for in-solution digestion, if enzymes perform poorlyfor in-gel digestion.

Liquid chromatography-multiple reaction monitoring mass spectrometry(LC-MRM) is used to screen for constant region and variable regionpeptides. Standards are then synthesized for the endogenous peptideusing stable isotope-labeled amino acids or single conservative aminoacid replacements and characterized by LC-MRM to test assay sensitivity(LOD, LLOQ, ULOQ), linearity, accuracy, precision, and other figures ofmerit.

For peptides with highly unique sequences but poor response in the massspectrometer, chemical labeling with tandem mass tags (TMT, ProteomeSciences) or tags for relative and absolute quantification (mTRAQ oriTRAQ, ABSciex) are applied to enhance the signal for those peptides. Asneeded, enrichment of the immunoglobulin light and heavy chains areachieved by SDS-PAGE, MW fractionation, or affinity pulldown.

Biomarkers are applied to patient samples to measure disease burden.LC-MRM is used to quantify constant region peptides to measure the totalclass of each immunoglobulin and its isoforms. The exact peptides dependon the enzyme selected. Examples for trypsin are provided on thefollowing pages. LC-MRM is also used to quantify variable regionpeptides to measure the disease-specific immunoglobulin. Alternatively,LC-MS/MS or pseudo reaction monitoring (PRM) strategies can also beused.

Quantification of the disease-specific peptides can be used to definepatients progressing from pre-malignant conditions, measure response totherapy, and detect relapse.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for monitoring a plasma cell dyscrasia in a subject,comprising: a) treating a biological sample comprising immunoglobulinfrom the subject to enzymatically cleave a target immunoglobulinassociated with the plasma cell dyscrasia into one or more variabledomain peptide fragments of the target immunoglobulin; and b) measuringthe one or more variable domain peptide fragments in the sample byquantitative mass spectrometry to quantify the amount of the targetimmunoglobulin in the sample; wherein the plasma cell dyscrasia ismonitored by the amount of the target immunoglobulin in the sample. 2.The method of claim 1, wherein the one or more variable domain peptidefragments are first identified by a method comprising: c) determiningthe amino acid sequence of the target immunoglobulin; and d) identifyingin silico one or more variable domain peptide fragments formed byenzymatic digestion of the target immunoglobulin that: i) contain aminoacid sequences from the variable domain of the target immunoglobulinthat are sufficiently unique to distinguish the target immunoglobulinfrom other immunoglobulin in the biological sample, and ii) are capableof being detected by a mass spectrometer.
 3. The method of claim 2,wherein the one or more variable domain peptides fragments contain thecomplementarity defining region (CDR) of the target immunoglobulin. 4.The method of claim 2, wherein the amino acid sequence of the targetimmunoglobulin is determined by a method comprising: e) RNA-sequencingimmunoglobulin mRNA from a plasma cell of the subject that is associatedwith the plasma cell dyscrasia; and f) in silico translating the RNAsequence of the immunoglobulin to protein.
 5. The method of claim 1,wherein the plasma cell dyscrasia comprises a multiple myeloma, whereinthe plasma cells are CD138⁺ cells from bone marrow aspirates of thesubject, wherein the method monitors tumor burden in the subject.
 6. Themethod of claim 1, further comprising measuring in the sample afterenzymatic cleavage one or more constant domain peptides fragmentscontaining amino acid sequences from a constant domain of theimmunoglobulin to quantify total immunoglobulin in the sample.
 7. Themethod of claim 6, wherein the plasma cell dyscrasia is monitored by theratio of target immunoglobulin to total immunoglobulin in the biologicalsample.
 8. The method of claim 1, wherein the target immunoglobulincomprises human heavy chain of IgG, IgA, IgM, IgD, or IgE.
 9. The methodof claim 1, wherein the target immunoglobulin comprises human kappalight chain or human lambda light chain.
 10. The method of claim 1,wherein the plasma cell dyscrasia is multiple myeloma or monoclonalgammopathy of undetermined significance (MGUS).
 11. The method of claim1, wherein the one or more peptide fragments are measured by spiking induring the mass spectrometry a known amount of the one or more peptidescontaining a specific label.
 12. The method of claim 11, wherein thespecific label comprises a heavy isotope label or an amino acidsubstitution sufficient to create a detectable mass difference.
 13. Themethod of claim 12, wherein the heavy isotope label is ²H, ¹³C, or ¹⁵N.14. The method of claim 1, wherein the immunoglobulin in the sample isdenatured prior to enzymatic cleavage.
 15. The method of claim 14,wherein the immunoglobulin is denatured by treatment with urea,disulfide reduction, and/or cysteine alkylation.
 16. The method of claim1, wherein the immunoglobulin in the sample is enzymatically cleaved byproteolytic enzyme digestion.
 17. The method of claim 16, wherein theproteolytic enzyme is trypsin.
 18. The method of claim 1, furthercomprising a treating step to isolate the target immunoglobulin prior toenzymatic cleavage, wherein the treating step comprises one or more ofsize exclusion chromatography, gel electrophoresis, and/or affinitychromatography.
 19. The method of claim 1, wherein the mass spectrometrycomprises liquid chromatography coupled to multiple reaction monitoring(LC-MRM).
 20. The method of claim 19, wherein the mass spectrometry isconducted on a triple quadrupole mass spectrometer.
 21. The method ofclaim 1, wherein the method is used to monitor for progression ofmonoclonal gammopathy of undetermined significance in the subject tomultiple myeloma or relapse of disease after prior treatment.
 22. Themethod of claim 1, wherein the method is used to monitor the efficacy ofa treatment regimen on a subject with multiple myeloma or MGUS.